Smad7 mRNA Expression Research Articles

The involvement of miR-6615-5p/Smad7 axis and immune imbalance in ammonia-caused inflammatory injury via NF-κB pathway in broiler kidneys

Ammonia (NH3), a toxic gas, has deleterious effects on chicken health in intensive poultry houses. MicroRNA can mediate inflammation. The complex molecular mechanisms underlying NH3 inhalation–caused inflammation in animal kidneys are still unknown. To explore the mechanisms, a broiler model of NH3 exposure was established. Kidney samples were collected on day 14, 28, and 42, and meat yield was evaluated on day 42. We performed histopathological examination, detected miR-6615-5p and mothers against decapentaplegic homolog 7 (Smad7), and determined inflammatory factors and cytokines in kidneys. The results showed that excess NH3 reduced breast weight and thigh weight, which indicated that excess NH3 impaired meat yield of broilers. Besides, kidney tissues displayed histopathological changes after NH3 exposure. Meanwhile, the increases of inducible nitric oxide synthase (iNOS) activity and nitric oxide content were obtained. The mRNA and protein expression of inflammatory factors, including nuclear factor-κB (NF-κB), cyclooxygenase-2, prostaglandin E synthases, and iNOS increased, indicating that NF-κB pathway was activated. T-helper (Th) 1 and regulatory T (Treg) cytokines were downregulated, whereas Th2 and Th17 cytokines were upregulated, suggesting the occurrence of Th1/Th2 and Treg/Th17 imbalances. In addition, we found that Smad7 was a target gene of miR-6615-5p in chickens. After NH3 exposure, miR-6615-5p expression was elevated, and Smad7 mRNA and protein expression were reduced. In summary, our results suggest that NH3 exposure negatively affected meat yield; and miR-6615/Smad7 axis and immune imbalance participated in NH3-induced inflammatory injury via the NF-κB pathway in broiler kidneys. This study is helpful to understand the mechanism of NH3-induced kidney injury and is meaningful to poultry health and breed aquatics.

The Effect of Alpha Mangostin on Epithelial-Mesenchymal Transition on Human Hepatocellular Carcinoma HepG2 Cells Surviving Sorafenib via TGF-β/Smad Pathways.

Purpose: This study was intended to find out the impact of alpha mangostin administration on the epithelial-mesenchymal transition (EMT) markers and TGF-β/Smad pathways in hepatocellular carcinoma Hep-G2 cells surviving sorafenib. Methods: Hepatocellular carcinoma HepG2 cells were treated with sorafenib 10 μM. Cells surviving sorafenib treatment (HepG2surv) were then treated vehicle, sorafenib, alpha mangostin, or combination of sorafenib and alpha mangostin. Afterward, cells were observed for their morphology with an inverted microscope and counted for cell viability. The concentrations of transforming growth factor (TGF)-β1 in a culture medium were examined using ELISA. The mRNA expressions of TGF-β1, TGF-β1-receptor, Smad3, Smad7, E-cadherin, and vimentin were evaluated using quantitative reverse transcriptase–polymerase chain reaction. The protein level of E-cadherin was also determined using western blot analysis. Results: Treatment of alpha mangostin and sorafenib caused a significant decrease in the viability of sorafenib-surviving HepG2 cells versus control (both groups with P <0.05). Our study found that alpha mangostin treatment increased the expressions of vimentin (P <0.001 versus control). In contrast, alpha mangostin treatment tends to decrease the expressions of Smad7 and E-cadherin (both with P >0.05). In line with our findings, the expressions of TGF-β1 and Smad3 are significantly upregulated after alpha mangostin administration (both with P <0.05) versus control. Conclusion: Alpha mangostin reduced cell viability of sorafenib-surviving HepG2 cells; however, it also enhanced epithelial–mesenchymal transition markers by activating TGF-β/Smad pathways.

SMAD4 Overexpression in Patients with Sleep Apnoea May Be Associated with Cardiometabolic Comorbidities.

Obstructive sleep apnoea (OSA) is associated with several diseases related to metabolic and cardiovascular risk. Although the mechanisms involved in the development of these disorders may vary, OSA patients frequently present an increase in transforming growth factor beta (TGFβ), the activity of which is higher still in patients with hypertension, diabetes or cardiovascular morbidity. Smad4 is a member of the small mother against decapentaplegic homologue (Smad) family of signal transducers and acts as a central mediator of TGFβ signalling pathways. In this study, we evaluate Smad4 protein and mRNA expression from 52 newly diagnosed OSA patients, with an apnoea–hypopnoea index (AHI) ≥30 and 26 healthy volunteers. These analyses reveal that OSA patients exhibit high levels of SMAD4 which correlates with variation in HIF1α, mTOR and circadian genes. Moreover, we associated high concentrations of Smad4 plasma protein with the presence of diabetes, dyslipidaemia and hypertension in these patients. Results suggest that increased levels of SMAD4, mediated by intermittent hypoxaemia and circadian rhythm deregulation, may be associated with cardiometabolic comorbidities in patients with sleep apnoea.

Tanshinone IIA alters the transforming growth factor-β1/Smads pathway in angiotensin II-treated rat hepatic stellate cells

ObjectiveTo investigate the effects of tanshinone IIA on the transforming growth factor-β1 (TGF-β1)/Smads signaling pathway in angiotensin II-treated hepatic stellate cells (HSCs).MethodsHSCs were cultured and treated with angiotensin II (10 μM) or angiotensin II (10 μM) plus tanshinone IIA (3, 10, or 30 μM). Cells were incubated for 48 hours and proliferation was determined with the Cell Counting Kit-8. The relative mRNA expression of TGF-β1, Smad4, and Smad7 was measured by quantitative real-time PCR, and the relative protein expression levels were investigated by western blotting.ResultsAfter angiotensin II treatment, cell proliferation was significantly accelerated. Furthermore, both the mRNA and protein expression of TGF-β1 and Smad4 was significantly up-regulated, while the mRNA and protein expression of Smad7 was significantly down-regulated compared with the control cells. Tanshinone IIA inhibited the observed effects of angiotensin II in a concentration-dependent manner, with significant inhibition exerted by tanshinone IIA at 10 and 30 μM.ConclusionsAngiotensin II promotes the proliferation of HSCs, possibly by regulating the expression of components along the TGF-β1/Smads signaling pathway. Tanshinone IIA inhibits the angiotensin II-induced activation of this pathway, and may, therefore, have preventive and therapeutic effects in liver fibrosis.

Mechanism of Anti-Hepatic Fibrosis of TGF-β1/Smad Signaling Pathway Affected by Chymase Inhibitors

Objective: This study aims to investigate the pathological mechanism of chymase inhibitors (Chy-I) on liver tissue in rats with hepatic fibrosis. Methods: Rats in the model group and chymase inhibitor group were established by using carbon tetrachloride. Rats in the chymase inhibitor group intragastrically received Chy-I (10 mg/kg/d) during the modeling. The pathological changes of rat liver tissues induced by chymase inhibitors were investigated, and the mRNA and protein expression of TGF-β1, Smad3 and Smad7 were observed in rat liver tissues. Results: When compared to the model group, The results presented that the liver tissue pathology of rats had been significantly improved in the Chy-I group. When compared to the normal group, the mRNA expression level and protein expression level of TGF-β1 and Smad3 in liver tissues had significantly increased, but the mRNA expression level and protein expression level of Smad7 significantly decreased (p &lt; 0 05). When compared to the model group, the mRNA expression level and protein expression level of TGF-β1 and Smad3 was significantly downregulated, but the mRNA expression level and protein expression level of Smad7 was significantly upregulated in the Chy-I group. Conclusion:Chy-I plays an active role in blocking hepatic fibrosis in rats by affecting the TGF-β1/Smad signaling pathway in liver tissues through multiple sites.

Deubiquitinase USP13 promotes extracellular matrix expression by stabilizing Smad4 in lung fibroblast cells

Smad4 plays a central role in the regulation of extracellular matrix (ECM) protein expression and cell differentiation; however, the molecular regulation of Smad4 protein stability by a deubiquitinase has not been reported. In the current study, we reveal that a deubiquitinase USP13 stabilizes Smad4, ultimately modulating ECM protein expression in lung fibroblast cells. USP13 was increased in primary adult lung fibroblasts isolated from bleomycin-challenged mice and transforming growth factor (TGF)-β1-treated primary mouse lung fibroblasts. In a bleomycin-induced murine model of lung fibrosis, USP13-deficientmiceshowed reduced ECM levels such as fibronectin (FN) and collagen compared with wild-type mice. The reductions in both protein levels and mRNA expression of ECM were observed in the isolated lung fibroblasts from USP13-deficient mice, suggesting that downregulation of USP13 reduces ECM levels through inhibiting its transcription. To investigate the molecular mechanisms by which USP13 modulates ECM expression, we focused on the role of USP13 on Smad4 expression. Overexpression of USP13 increased FN and Smad4 protein levels in lung fibroblasts, while downregulation ofUSP13 reduced Smad4 protein levels, without altering Smad4 mRNA expression, suggesting that USP13 regulates Smad4 protein stability. Knockdown of USP13 decreased Smad4 half-life and promoted Smad4 ubiquitination. Both Smad4 and USP13 were co-localized in the cytoplasm in treated cell, and co-translocated into the nucleus in response to TGF-β1.The results indicate that USP13 promotes ECM expression by stabilizing Smad4 in lung fibroblasts and plays a role in the maintenance of the extracellular matrix in lungs.

Synovial and pulmonary dysfunctions are induced by crosstalk of Smad and Erk pathways in an arthritis model

In the current experiment, the effects of transforming growth factor (TGF)-β1/Smad and ERK pathway crosstalk on synovial and pulmonary systems during rheumatoid arthritis have been investigated. For this purpose, rats were divided into normal control (NC) and model control (MC) groups. In the MC group, 0.1 ml Freund's complete adjuvant was injected intradermally into the right hind paw, and the resulting inflammation represented a rheumatoid arthritis model. Joint swelling and changes in lung functions were observed in arthritic rats. Synovial and lung were observed by light and electron microscopies. Enzyme-linked immunosorbent assays were used to detect TGF-β1, interleukin (IL)-1β, IL-4, IL-10, interferon-γ (IFN-γ), connective tissue growth factor (CTGF), and fibroblast growth factor (FGF). PCR, immunohistochemistry, and immunoblotting were used to detect changes in Smad and ERK pathways of synovial and lung tissues. Compared with the NC group, toe swelling was elevated in the MC group. Pulmonary functions FEV1, FEF50, FEF75, MMF, and PEF were decreased (P&lt; 0.01). Serum cytokines IL-1β, IL-4, TGF-β1, and CTGF were increased, while IFN-γ, IL-10, Th1/Th2 cell ratio, and FGF were decreased (P&lt; 0.01 or P&lt; 0.05). Expression of TGF-β1 and Smad2/3/4 mRNAs and TGF-β1, TβRI, TβRII, Smad2/3, p-Smad2/3, and Smad4 proteins in the synovial membrane and lung tissue were increased, and expression of Smad7 mRNA and protein was decreased (P&lt;0.01) or P&lt;0.05). Expression of ERK2 mRNA and p-ERK1/2 protein was increased in the synovial membrane and lung tissue, and expression of ERK1/2 mRNAs and ERK1/2 and p-ERK1/2 proteins was increased in lung tissue (P&lt; 0.01 or P&lt; 0.05). Correlation analysis showed that FEV1 was negatively correlated with TGF-β1 mRNA and protein in arthritic rats, FEF25 was negatively correlated with Smad4 protein, and FEF50 was negatively correlated with the TβRII protein, and FEF75, TGF-β1 and Smad3 mRNAs. There was a negative correlation between Smad2/3 protein and a negative correlation between PEF and TGF-β1 protein (P&lt; 0.05). FEF50 and MMF were positively correlated with Smad7 mRNA (P&lt; 0.05). FEV1 was negatively correlated with ERK2 mRNA, and FEF25 was negatively correlated with p-ERK1/2 protein. FEF75 and MMF were negatively correlated with ERK1/2 and p-ERK1/2, respectively (P&lt; 0.05). ERK1 mRNA was positively correlated with Smad3 mRNA and TβRII protein, ERK2 mRNA was positively correlated with p-Smad2/3, and ERK1/2 protein was positively correlated with Smad2 mRNA, Smad4 protein, p-ERK1/2 protein, Smad4 mRNA, and p-Smad2/3 protein (P&lt; 0.05). p-ERK1/2 protein was negatively correlated with Smad7 protein (P&lt; 0.05). It is concluded that arthritic rats have synovial and systemic pulmonary damage. Smad and ERK pathway crosstalk leads to systemic lesions. Smad and ERK pathways are gradually activated by phosphorylation under the induction of the TGF-β1 promoter, and then participate in transcriptional activities, leading to the increase in synovial inflammation of arthritis, pulmonary lesions, and decreases in lung functions.

MiR-146a-5p Regulated Cell Proliferation and Apoptosis by Targeting SMAD3 and SMAD4.

Background: microRNAs (miRNAs) are a small, endogenous non-coding RNAs that are involved in post-transcriptional gene regulation of many biological processes, including embryo implantation and placental development. In our previous study, miR-146a-5p was found expressed higher in the serum exosomes of pregnant sows than non-pregnant. The research on miR-146a-5p has been mainly related to human diseases, but there are few studies on its effects on the reproduction of sows in early pregnancy.Objective: In this article, our motivation is to study the role of miR-146a-5p in the early pregnancy of sows on the cell proliferetion and apoptosis by targeting SMAD3 and SMAD4.Methods: Bioinformatics software was used to identify the target genes of miR-146a-5p. The wild-type and mutant-type recombinant plasmids of dual-luciferase reporter with 3'-UTR of Smad3 or 3'-UTR of Smad4 were constructed, and co-transfected in porcine kidney cell (PK-15 cell) with miR-146a-5p mimic, mimic-NC(M-NC), inhibitor and inhibitor-NC(IN-NC), then dual-luciferase activity analysis, qRT-PCR and Western blot were performed to verify the target genes. After the transfection of BeWo choriocarcinoma cell (BeWo cell) with miR-146a-5p mimic, M-NC, inhibitor and IN-NC, the mRNA expression of Caspase-3, BAX and Bcl-2 was measured using qRT-PCR, and the cell proliferation was measured using CCK-8 kit.Results: The luciferase, mRNA and protein expression of Smad3 in PK-15 cells treated by Smad3-3'-UTR-W co-transfected with miR-146a-5p mimic were significantly lower than that with miR-146a-5p M-NC, and the results of Smad4 were similar to Smad3, but the protein expression had a trend to lower in mimic group. The expression level of Bcl-2 in the miR-146a-5p mimic group was significantly lower than that in the miR-146a-5p M-NC group, but the expression pattern of Caspase-3 was just opposite. The mimic of miR-146a-5p reduced the proliferation of BeWo cells, however the inhibitor increased.Conclusion: Smad3 and Smad4 are the direct target genes of miR-146a-5p. The expression of Smad3 and Smad4 were affected by the mimic and inhibitor of miR-146a-5p. miR-146a-5p affects cell apoptosis and proliferation by regulating their target genes. This study provided new data to understand the regulation mechanism of early pregnancy in sows.

Inhibiting of self-renewal, migration and invasion of ovarian cancer stem cells by blocking TGF-β pathway.

To investigate the effect and mechanism of SB525334 on self-renewal, migration and invasion of ovarian cancer stem cells. ALDHhigh-expressing cancer stem cells (CSCs) were isolated from human ovarian cancer cell line SKOV-3 by flow cytometry and treated with 2μg/mL SB525334 for 6h. The sphere forming assay was used to detect the ability of self-renewal of CSCs and the colony formation assay was used to detect the tumorigenicity in vitro. Transwell migration and invasion assay were used to detect the migration and invasion ability of CSCs. To further explore the mechanism, real-time quantitative PCR and flow cytometry were used to detect the mRNA and protein expression of TGF-β, Smad2, Smad3, phosphorylated Smad2, phosphorylated Smad3 and Smad4, respectively. Expressions of epithelial-mesenchymal transition (EMT)-related genes E-cadherin, Snail, Vimentin were also assessed. The self-renewal ability, tumorigenicity in vitro, migration and invasion ability of CSCs were significantly attenuated after SB525334 treatment. The expressions of TGF-β, phosphorylated Smad2, phosphorylated Smad3, Snail, and Vimentin were decreased, while Smad4 and E-cadherin expressions were increased. SB525334 may inhibit the self-renewal, invasion and migration of ovarian CSCs by blocking the TGF-β/Smad/EMT pathway.

Differential Expression of SMAD Genes and S1PR1 on Circulating CD4+ T Cells in Multiple Sclerosis and Crohn's Disease.

The Th17 immune response plays a key role in autoimmune diseases such as multiple sclerosis (MS) and inflammatory bowel disease (IBD). Expression of Th17-related genes in inflamed tissues has been reported in autoimmune diseases. However, values are frequently obtained using invasive methods. We aimed to identify biomarkers of MS in an accessible sample, such as blood, by quantifying the relative expression of 91 Th17-related genes in CD4+ T lymphocytes from patients with MS during a relapse or during a remitting phase. We also compared our findings with those of healthy controls. After confirmation in a validation cohort, expression of SMAD7 and S1PR1 mRNAs was decreased in remitting disease (–2.3-fold and –1.3-fold, respectively) and relapsing disease (–2.2-fold and –1.3-fold, respectively). No differential expression was observed for other SMAD7-related genes, namely, SMAD2, SMAD3, and SMAD4. Under-regulation of SMAD7 and S1PR1 was also observed in another autoimmune disease, Crohn’s disease (CD) (−4.6-fold, -1.6-fold, respectively), suggesting the presence of common markers for autoimmune diseases. In addition, expression of TNF, SMAD2, SMAD3, and SMAD4 were also decreased in CD (–2.2-fold, –1.4-fold, –1.6-fold, and –1.6-fold, respectively). Our study suggests that expression of SMAD7 and S1PR1 mRNA in blood samples are markers for MS and CD, and TNF, SMAD2, SMAD3, and SMAD4 for CD. These genes could prove useful as markers of autoimmune diseases, thus obviating the need for invasive methods.

Characteristics of TGFBR1–EGFR–CTNNB1–CDH1 Signaling Axis in Wnt-Regulated Invasion and Migration in Lung Cancer

This study aimed to explore the characteristics of TGFBR1–epidermal growth factor receptor (EGFR)–CTNNB1–CDH1 axis in regulating the invasion and migration in lung cancer. Using the small interfering RNA technology, EGFR was silenced in H2170 and H1299 cells. Then, the colony formation, migration, and invasion abilities were detected using colony-forming assay and transwell assay. Moreover, the mRNA expression of smad2, smad3, CTNNB1, and CDH1, and the protein expression of TGFBR1, CDH1, and TCF were determined using the real-time polymerase chain reaction and western blotting. The results showed that silencing EGFR could significantly decrease the colony-forming ability in H2170 and H1299. Knocking down EGFR could significantly inhibit the invasion and migration ability of H2179 and H1299. Inhibiting the expression of EGFR could significantly decrease the expression of smad2, smad3, CDH1, and CTNNB1, with all P-values <0.05. In addition, silencing EGFR could markedly decrease the expression of TGFBR1 and CDH1 in H1299 and H2170, with all P-values <0.05. In conclusion, silencing EGFR could significantly regulate the progression of lung cancer via TGFBR1–EGFR–CTNNB1–CDH1 axis in Wnt signaling pathway.

Renoprotective effect of vildagliptin following hepatic ischemia/reperfusion injury

Remote renal injury is a drastic consequence of hepatic ischemia/reperfusion (IR) injury. Vildagliptin (V) is a dipeptidyl peptidase-4 inhibitor that has a hepatorenal protective effect against models of liver and renal IR. This research was done to explore the protective role of vildagliptin against renal injury following hepatic IR injury as well as the possible involvement of transforming growth factor-beta (TGF-β)/Smad/alpha-smooth muscle actin (α-SMA) expressions in the pathophysiological mechanism of the remote renal injury. Three groups of male Wistar rats were organized into: sham group, IR group, and V + IR group in which 10 mg/kg/day of vildagliptin was pretreated for 10 days intraperitoneally. Blood in addition to renal and hepatic tissue samples was used for biochemical and histopathological studies. Hepatic IR induced a marked increase in serum creatinine, blood urea nitrogen, liver enzymes, renal nitric oxide, malondialdehyde, tumor necrosis factor-alpha levels with a marked upregulation of renal mRNA expressions of TGF-β, Smad2, Smad3, and α-SMA in addition to a marked decline in renal catalase content comparing to the sham group. Abnormal histopathological findings of hepatic and renal injury were detected in the IR group. Vildagliptin significantly improved these biochemical markers as well as the histopathological changes. The upregulation of renal TGF-β/Smad/α-SMA mRNA expressions was involved for the first time in the pathogenesis of the renal injury following hepatic IR and vildagliptin ameliorated this renal injury through blocking these expressions.

Canonical Signaling By TGF Family Members in Mesenchymal Stromal Cells Is Dispensable for Hematopoietic Niche Maintenance Under Basal and Stress Conditions

The bone marrow contains a complex population of stromal and hematopoietic cells that together generate a unique microenvironment, or niche, to support hematopoiesis. Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche and include CXCL12-abundant reticular (CAR) cells, adipocytes, osteolineage cells, and arteriolar pericytes, all of which have been implicated in hematopoietic stem/progenitor cell (HSPC) maintenance. There also is evidence that adaptive changes in bone marrow stromal cells contributes to recovery from myelosuppresive therapy and the development of certain hematopoietic malignancies. However, the signals that contribute to the development, maintenance, and stress response of bone marrow mesenchymal stromal cells are poorly understood. Here, we test the hypothesis that cytokines of the transforming growth factor superfamily, which include bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), and activins/inhibins, provide signals to mesenchymal stromal cells that contribute to basal and stress hematopoiesis responses. To test this hypothesis, we abrogated canonical TGF family signaling in mesenchymal stem/progenitor cells by deleting Smad4 using a doxycycline-repressible Osterix-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow. We first performed lineage-tracing studies using Osx-Cre Smad4fl/fl Ai9 mice to show that activation of Osx-Cre at birth (by removal of doxycycline) results in the efficient targeting of bone marrow mesenchymal stromal cells. Moreover, we show that Smad4 mRNA expression is essentially undetectable in sorted mesenchymal stromal cells sorted from the bone marrow of these mice. Basal hematopoiesis and bone marrow stromal cells were analyzed in 6-8 week old Osx-Cre Smad4fl/fl mice. No alterations in the number or spatial organization of CAR cells, osteoblasts, or adipocytes was observed, and expression of key niche factors, including Scf, Cxcl12, and Spp1 was normal. Basal hematopoiesis, including the number of phenotypic HSCs in bone marrow and spleen, also was normal. Recent studies have shown that inhibition of activin signaling by treating with an activin receptor 2 alpha (ACVR2a) ligand trap stimulates erythropoiesis. Although ACVR2a signaling in erythroid progenitors contributes to this effect, two groups showed that inhibition of ACVR2a signaling in bone marrow stromal cells also stimulates erythropoiesis. Thus, we next characterized basal and stress erythropoiesis in Osx-Cre Smad4fl/fl mice. The frequency of phenotypic erythroid progenitors in bone marrow and spleen was similar to control mice. The stress erythropoiesis response was assessed after induction of acute hemolytic anemia by phenylhydrazine treatment. Both the magnitude of anemia and kinetics of erythroid recovery were similar to control mice. Myelosuppressive therapy induces marked alterations in the bone marrow microenvironment that includes an expansion of osteolineage cells and adipocytes, which have been linked to hematopoietic recovery. Thus, we next characterized stress hematopoiesis in Osx-Cre Smad4fl/fl mice in response to 5-fluorouracil (5-FU) treatment. Compared to control mice, the magnitude and duration of neutropenia following 5-FU were similar. Moreover, mouse survival after repeated weekly doses of 5-FU was comparable to control mice. HSPC mobilization by G-CSF is due, in large part, by downregulation of CXCL12 expression in bone marrow mesenchymal stromal cells. A prior study suggested that SMAD signaling negatively regulates CXCL12 expression in stromal cells. Consistent with this finding, we show that treatment of cultured bone marrow derived MSCs with TGF-b1 for 48 hours results in a significant (3.3-fold, P&lt;0.0001) decrease in CXCL12 mRNA expression. Thus, in the final experiments, we characterized G-CSF induced HSPC mobilization in Osx-Cre, Smad4fl/fl or Osx-Cre, Tgfbr2fl/fl mice. HSPC mobilization, as quantified by CFU-C and Kit+ Sca+ lineage- (KSL) cell number in blood or spleen after 5 days of G-CSF treatment was comparable to control mice. Collectively, these data suggest the TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions. Disclosures No relevant conflicts of interest to declare.

Extract of Averrhoacarambola L. (Oxalidaceae) roots ameliorates carbon tetrachloride-induced hepatic fibrosis in rats

Ethnopharmacological relevanceThe root of Averrhoa carambola L. (Oxalidaceae), a traditional Chinese medicine, was mainly used in ancient times in the treatment of urinary calculi, recurrent headache and joint pain. Aim of the studyOur aims were to explore the potential therapeutic effect of the extract of Averrhoa carambola L. (Oxalidaceae) roots (EACR) against hepatic fibrosis in CCl4-treated rats and to understand the underlying molecular mechanism. Materials and methodsSix groups of male Sprague Dawley rats were treated as follows: vehicle (olive oil), CCl4 alone, CCl4+colchicine, CCl4+EACR 1.0 g/kg, CCl4+EACR 0.5 g/kg and CCl4+EACR 0.25 g/kg. At the end of the 12th week, biomarkers of liver function, liver fibrosis, hepatic oxidative stress and antioxidant status were assayed, and histopathological and immunohistochemical evaluation of liver tissue were conducted to investigate the liver damage and fibrosis degree. Furthermore, expressions of COL-1a1, α-SMA, TGF-β1, Smad2, smad3, Smad4 and TIMP2 were examined by qPCR and/or western blot. The expressions of apoptosis-related proteins were also detected using western blot analysis. ResultsEACR treatment markedly reduced the CCl4-induced elevation of serum aminotransferase activities, liver fibrosis indexes, and the extent of oxidative stress. EACR treatment also significantly reduced the accumulation of collagen and the immunostaining of α-SMA, TGF-β1 and Smad2, 4 and 7 in the liver of CCl4 treated rats. In addition, EACR treatment markedly reversed the CCl4-induced increase in mRNA expression of COL-1a1, α-SMA, TIMP2, TGF-β1, Smad2 and Smad4 and suppressed the expressions of α-SMA, TIMP2, TGF-β1, smad2, 3 and 4, BAX and cleaved caspase-3 proteins. Meanwhile, EACR treatment also significantly elevated the mRNA expression of Smad7 and the protein expression of Smad7 and Bcl-2. ConclusionThese results suggest that EACR has protective activity against liver fibrosis. The anti-fibrotic activity of EACR in vivo is associated with enhanced antioxidant, apoptosis-inhibition and increased MMP-2/TIMP-2 expression ratio, and with modulation of TGF-β1/Smad signaling pathway.

Effects of Bone Marrow-Derived Mesenchymal Stem Cells on Hypoxia and the Transforming Growth Factor beta 1 (TGFβ-1) and SMADs Pathway in a Mouse Model of Cirrhosis.

BackgroundThe role of bone marrow-derived mesenchymal stem cells (BM-MSCs) in liver fibrosis remains poorly understood. This study aimed to use a mouse model of carbon tetrachloride (CCL4)-induced liver fibrosis to investigate the effects of BM-MSCs during liver hypoxia and the involvement of the transforming growth factor beta 1 (TGF-β1) and SMADs pathway.Material/MethodsThirty C57BL/6 mice were randomly divided into the control group (n=10), the model group (n=10), and the BM-MSC-treated model group (n=10). In the model group, liver fibrosis was induced by intraperitoneal injection of CCl4. BM-MSCs were transplanted after 12 weeks of CCl4 treatment. The serum biochemical parameters and histological changes in the liver, using histochemical stains, were investigated. The expression of collagen type I (collagen I), alpha-smooth muscle actin (α-SMA), TGF-β1, SMAD3, SMAD7, hypoxia-inducible factor 1 alpha (HIF-1α), and vascular endothelial grow factor (VEGF) were assessed by immunohistochemistry and quantitative real-time polymerase chain (RT-qPCR) reaction.ResultsTreatment with BM-MSCs reduced the expression of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared with the model group, and reduced liver fibrosis determined histologically using hematoxylin and eosin (H&E) and Masson’s trichrome staining compared with the model group. The area of liver fibrosis decreased after BM-MSCs treatment (p<0.05). Protein expression of HIF-1α and VEGF were decreased after BM-MSCs treatment (p<0.05). Transplantation of BM-MSCs reduced the mRNA expression of TGF-β1, collagen I, α-SMA, and SMAD3 (p<0.05).ConclusionsBM-MSC transplantation reduced CCl4-induced murine liver fibrosis, indicating that in a hypoxic microenvironment, BM-MSCs may inhibit the TGFβ-1/SMADs pathway.

Hesperidin ameliorates bleomycin-induced experimental pulmonary fibrosis via inhibition of TGF-beta1/Smad3/AMPK and IkappaBalpha/NF-kappaB pathways.

Bleomycin (BLM) is a chemotherapeutic agent which is associated with Idiopathic pulmonary fibrosis (IPF) due to its chronic administration. Hesperidin, a bioflavonoid has been reported to possess antioxidant, anti-inflammatory, wound healing, and antiapoptotic potential. To evaluate the therapeutic potential of hesperidin against BLM-induced pulmonary fibrosis and decipher its possible mechanism of action. Intraperitoneal administration of BLM (6 IU/kg) caused induction of IPF in Sprague-Dawley rats. Rats were treated with hesperidin (25, 50, and 100 mg/kg, p.o.) for 28 days, followed by estimation of various parameters in bronchoalveolar lavage fluid (BALF) and lung. Hesperidin (50 and 100 mg/kg) administration significantly ameliorated (p < 0.05) alterations induced by BLM in lung index, percent oxygen saturation, serum ALP and LDH levels, BALF differential cell count, and lung function test. Elevated levels of oxido-nitrosative stress, hydroxyproline, and myeloperoxidase levels in BALF and lung were significantly decreased by hesperidin on day 14. Hesperidin significantly inhibited BLM-induced down-regulated lung Nrf2 and HO-1 as well as up-regulated TNF-α, IL-1β, IL-6, collagen-1, TGF-β, and Smad-3 mRNA expressions. Western blot analysis showed that alteration in lung NF-κB, IκBα, AMPK, and PP2C-α protein expressions were ameliorated by hesperidin on day 28. Furthermore, BLM induced histological and ultrastructural aberrations in the lung which were attenuated by hesperidin treatment. Hesperidin alleviates BLM-induced IPF via inhibition of TGF-β1/Smad3/AMPK and IκBα/NF-κB pathways which in turn ameliorate the modulation of oxido-inflammatory markers (Nrf2 and HO-1) and pro-inflammatory markers (TNF-α, IL-1β, and IL-6) to reduce collagen deposition during pulmonary fibrosis. See also Figure 1(Fig. 1).

Influence of Ceramic Debris on Osteoblast Behaviors: AnIn VivoStudy

ObjectiveWear‐induced aseptic loosening has been accepted as one of the main reasons for failure of total hip arthroplasty. Ceramic wear debris is generated following prosthesis implantation and plays an important part in the upregulation of inflammatory factors in total hip arthroplasty. The present study investigates the influence of ceramic debris on osteoblasts and inflammatory factors.MethodsCeramic debris was prepared by mechanical grinding of an aluminum femoral head and added to cultures of MC3T3‐E subclone 14 cells at different concentrations (i.e. 0, 5, 10, and 15 μg/mL). Cell proliferation was evaluated using a Cell Counting Kit (CCK‐8), and cell differentiation was assessed by mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In addition, cell bio‐mineralization was evaluated through alizarin red S staining, and release of tumor necrosis factor alpha (TNF‐α), interleukin‐1 beta (IL‐1β), and interleukin‐6 (IL‐6) was measured through enzyme‐linked immunosorbent assays (ELISA). Furthermore, mRNA expression of Smad1, Smad4, and Smad5 and protein expression of phosphorylated Smad1, Smad4, and Smad5 were measured by reverse transcriptase polymerase chain reaction (RT‐PCR) and western blotting.ResultsThe ceramic debris had irregular shapes and sizes, and analysis of the size distribution using a particle size analyzer indicated that approximately 90% of the ceramic debris was smaller than 3.2 μm (2.0 ± 0.4 μm), which is considered clinically relevant. The results for mRNA expression of ALP, OCN, and OPN and alizarin red S staining indicated that cell differentiation and bio‐mineralization were significantly inhibited by the presence of ceramic debris at all tested concentrations (P < 0.05, and the values decreased gradually with the increase of ceramic debris concentration), but the results of the CCK‐8 assay showed that cell proliferation was not significantly affected (P > 0.05; there was no significant difference between the groups at 1, 3, and 5 days). In addition, the results of ELISA, RT‐PCR, and western blotting demonstrated that ceramic debris significantly promoted the release of inflammatory factors, including TNF‐α, IL‐β, and IL‐6 (P < 0.05, and the values increased gradually with the increase of ceramic debris concentration), and also greatly reduced the mRNA expression of Smad1, Smad4, and Smad5 (the values decreased gradually with the increase of ceramic debris concentration) as well as protein expression of phosphorylated Smad1, Smad4, and Smad5.ConclusionCeramic debris may affect differentiation and bio‐mineralization of MC3T3‐E subclone 14 cells through the bone morphogenetic protein/Smad signaling pathway.

Astragaloside IV inhibits excessive mesangial cell proliferation and renal fibrosis caused by diabetic nephropathy via modulation of the TGF‑β1/Smad/miR‑192 signaling pathway

Astragaloside IV (ASI) exhibits a wide variety of pharmacological effects in cardiovascular diseases, hepatitis and kidney disease and due to this, ASI has recently become an attractive research target. The present study aimed to determine the effect of ASI on renal fibrosis and the mechanisms underlying its therapeutic effects in diabetic nephropathy (DN). In vitro, ASI was added to rat mesangial cells (RMCs) and cultured with a high level of glucose (HG) to observe the effects exhibited on proliferation and fibrosis-related mRNA and protein expression. In vivo, a DN model was established using streptozotocin administration in rats, and renal injury was evaluated using renal histological examination. The expression levels of related mRNAs and proteins were analyzed using reverse transcription-quantitative PCR, western blot analysis and immunohistochemistry. ASI was demonstrated to downregulate miR-192 expression and inhibit excessive proliferation of RMCs, which was induced by HG, in a dose-dependent manner. Additionally, ASI exhibited a therapeutic effect on DN rats. ASI was also demonstrated to decrease the miR-192 expression and mRNA and protein expression of transforming growth factor-β1 (TGF-β1), Smad3, α-smooth muscle actin (α-SMA) and collagen type 1 (col1), and increase the mRNA and protein expression of Smad7 in vitro and in vivo. These results suggested that ASI exhibited a therapeutic effect on DN, possibly due to the inhibition of excessive mesangial proliferation and renal fibrosis via the TGF-β1/Smad/miR-192 signaling pathway.

Effect of paeoniflorin on cardiac remodeling in chronic heart failure rats through the transforming growth factor β1/Smad signaling pathway.

Cardiac remodeling is an important mechanism for the occurrence and development of chronic heart failure (CHF). Paeoniflorin (Pae) is the main active ingredient of Chinese herbaceous peony and has novel anti-inflammatory effect. This study was conducted to assess the effects and mechanisms of Pae on cardiac remodeling in CHF rats. A cardiac remodeling rat model was induced by isoprenaline (Iso). Pae (20 µg/kg/d) was administrated to CHF rats for six weeks. Cardiac ultrasound was used to assess the structure and function of CHF rats. Collagen volume fraction (CVF) and perivascular collagen volume area of myocardial tissues were calculated. With real-time polymerase chain reaction and Western blot, the protein and mRNA levels of transforming growth factor β1 (TGF-β1) and Smad3 were detected. Compared to Iso group, Pae can alleviate cardiac remodeling and improve cardiac function in CHF rats. The levels of CVF and perivascular collagen volume area reduced in Pae group (P<0.05). The expression of TGF-β1 and Smad3 protein decreased in Pae and Cap group (P<0.05). Further, the expression of TGF-β1 and Smad3 mRNA also decreased markedly in the Pae group (P<0.05). Pae could attenuate cardiac remodeling and improve cardiac function in CHF rats. The potential mechanism for the cardioprotective effect of Pae may be highly associated with the down-regulating of TGF-β1/Smad signaling pathway.

Corrigendum] Human peripheral blood‑derived exosomes for microRNA delivery.

Exosomes serve important functions in cell‑to‑cell communication and biological functions by serving as a delivery cargo shuttle for various molecules. The application of an improved delivery method for microRNAs (miRNAs/miRs) may enhance their potential as a therapeutic tool in cardiac diseases. Thus, the present study investigated whether human peripheral blood‑derived exosomes may be used as a delivery cargo system for miRNAs, and whether the delivery of miR‑21 using a human peripheral blood derived‑exosome may influence the degree of remodeling following myocardial infarction (MI). In H9C2 and HL‑1 cells, miR‑21 expression was successfully regulated by treatment with human peripheral blood derived‑exosomes loaded with an miR‑21 mimic or inhibitor compared with untreated cells. In addition, the mRNA and protein expression levels of SMAD family member7 (Smad7), phosphatase and tensin homolog (PTEN) and matrix metalloproteinase 2 (MMP2), which are involved in cardiac fibrosis, were associated with the uptake of miR‑21 mimic‑ or inhibitor‑loaded exosomes. Similarly, the invivo mRNA and protein expression of Smad7, PTEN and MMP2 were altered following treatment with miR‑21 mimic‑ or inhibitor‑loaded exosomes. Furthermore, miR‑21 mimic‑loaded exosomes enhanced fibrosis, whereas miR‑21 inhibitor‑loaded exosomes reduced fibrosis in a mouse MI model. These results suggested that miRNA‑loaded human peripheral blood derived‑exosomes may be used as a therapeutic tool for cardiac diseases. [the original article was published in International Journal of Molecular Medicine 43: 2319‑2328, 2019; DOI:10.3892/ijmm.2019.4150].