TGF-β1 mRNA Expression Research Articles

Effect of Quercetin on the fructose-activated human hepatic stellate cells, LX-2, an in-vitro study.

Hepatic fibrosis is one of the main reasons for mortality in the world. Hepatic stellate cells (HSCs) activate during chronic liver injury, express more Transforming growth factor beta(TGF-β), Collagen1α (COLA1) and actin-alpha smooth muscle (αSMA) that lead to hepatic fibrosis. Quercetin is a flavonoid in vegetables and fruits that has shown hepatoprotective potential, but little is known about its effects on HSCs activation. In this study, we investigated the antifibrotic activity of Quercetin on fructose-activated human HSCs and its underlying mechanism in vitro. First, the human HSCs were treated with fructose (25mM) for 48h and then with Quercetin for 24h. Total RNAs were extracted, reversely transcribed into cDNA, Quantitative Real-time PCR and western blot were performed. The results showed that the levels of mRNA expression of TGF-β, αSMA, Collagen1 genes, and phosphorylated smad3 protein were significantly reduced in fructose-activated HSCs after treatment with Quercetin compared to fructose-activated HSCs. Quercetin is effective in reducing the expression of fibrogenic genes in fructose-activated human HSCs through downregulation of the TGF-β/smad3 signaling pathway. Therefore, Quercetin possesses significant antifibrotic properties in hepatic fibrosis.

P051 IFNγ-macrophages could mediate EMT in Crohn’s disease through the WNT pathway

Abstract Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. Methods The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT) through the WNT pathway. The mRNA and protein expression of IFNγ in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (10 ng/ml) for 4 days, the mRNA expression of WNT2b, WNT6 and TGFβ were determined by RT-PCR and protein. IFNγ-U937 were coculture with HT29 cells for 3 days and the expression of EMT markers, βCATENIN and WNT2b in HT29 cells were analyzed by WB. In some cases, HT29 cells were treated with the inhibitor of the WNT-pathway, XAV939 (1 μM), or were transfected with vectors-targeting human FZD4 (miFzd4). Results are expressed as mean±SEM. Statistical analysis was performed by ANOVA + Newman-Keuls or unpaired t-test. Results The mRNA and protein expression of IFNγ were significantly higher in intestinal samples from B2 CD patients (11.4±1.6 fold induction and 70,0 ± 2,3 pg/mg, respectively) and B3 CD patients (14.2±1.8 fold induction and 80,4 ± 6,8 pg/mg, respectively) than in controls (1,0±0,1 fold induction and 18,9 ± 0,3 pg/mg, respectively). U937 cells treated with IFNγ increased significantly the protein expression of WNT2b (1,3 ± 0,07 N=13* vs vehicle) but not the expression of WNT6 or TGFβ. IFNγ-U937 co-cultured with HT29 increased significantly the protein expression of EMT markers, βCATENIN and WNT2b in HT29 cells (VIMENTIN: 2,7 ± 0,3 N=6* vs vehicle-HT29; SNAIL1: 1,5 ± 0,1 N=5* vs vehicle-HT29; βCATENIN: 1,4 ± 0,09 N=5* vs vehicle-HT29; WNT2b: 2,0 ± 0,3 N=8* vs vehicle-HT29). Treatment HT29 cells with XAV939 (VIMENTIN: 1,4 ± 0,4 N=6 vs vehicle-HT29 XAV; SNAIL1: 1,0 ± 0,1 N=5 vs vehicle-HT29 XAV; βCATENIN: 0,8 ± 0,1 N=5 vs vehicle-HT29 XAV) or with miFzd4 (VIMENTIN: 0,3 ± 0,07 N=12* vs IFNγ-HT29 mock; SNAIL1: 0,5 ± 0,06 N=12* vs IFNγ-HT29 mock; βCATENIN: 0,6 ± 0,04 N=12* vs IFNγ-HT29 mock; FZD4: 0,5 ± 0,04 N=12* vs IFNγ-HT29 mock) partially reversed the increases produced by IFNγ-U937 in HT29 cells. Conclusion IFNγ-macrophages may stimulate the expression of WNT ligands in an autocrine and paracrine manner. The expression of WNT ligands at the epithelial level could favor mesenchymal epithelial transition through WNT2b ligand and FZD4 receptor.

Aerobic Exercise Improves Pulmonary Fibrosis by Improving Insulin Resistance and Inflammation in Obese Mice.

BackgroundPrevious studies have demonstrated that obesity is associated with pulmonary fibrosis. We attempted to identify whether regular aerobic exercise (AE) can protect against high-fat diet (HFD)-associated pulmonary fibrosis.MethodsForty-eight C57BL/6 mice were randomly assigned to four groups: chow group (Ch), chow plus exercise group (CE), obesity group (Ob), and obesity plus exercise group (OE). The mice were fed either an HFD or a chow diet for 16 weeks, and low-intensity aerobic exercise (AE) was performed in the last 8 weeks. We measured the degree of pulmonary fibrosis; pulmonary inflammation; oxidative stress parameters; insulin resistance-related indicators; the number of inflammatory cells in bronchoalveolar lavage fluid (BALF); the mRNA expression levels of IL-10, IL-1β, TGF-β, TNF-α, CXCL-1, IL-17, MMP-9, MPO, NE, and sirt-1; and the BALF levels of CXCL-1, IL-17, TGF-β, IL-10, IL-1β, and TNF-α in lung tissue.ResultsAE in obese mice protected against obesity-associated pulmonary fibrosis, chronic inflammation, pro-oxidative/antioxidative imbalance, and insulin resistance. AE ameliorated the HFD-induced inflammatory response and neutrophil infiltration in the lung. AE downregulated BALF levels of CXCL-1, IL-1β, TNF-α IL-17, and TGF-β but upregulated BALF levels of IL-10. AE decreased IL-1β, TGF-β, TNF-α, CXCL-1, IL-17, MMP-9, MPO, and NE mRNA expression levels but upregulated IL-10 and sirt-1 mRNA expression levels in the lung.ConclusionsAE protects against HFD-induced pulmonary fibrosis by improving obesity-associated insulin resistance, chronic low-grade inflammation, and pro-oxidative/antioxidative imbalance. AE improved HFD-induced pulmonary fibrosis by suppressing IL-17, TGF-β, NE, and MMP-9 expression and activating IL-10 and sirt-1 expression.

Periodontal ligament cells under mechanical force regulate local immune homeostasis by modulating Th17/Treg cell differentiation.

Improper orthodontic force often causes root resorption or destructive bone resorption. There is evidence that T helper 17 (Th17) cells and regulatory T (Treg) cells may be actively involved in bone remodeling during tooth movement. In a combination of in vitro and in vivo studies, we investigated the effect of human periodontal ligament cells (hPDLCs) on Th17/Treg cells under different orthodontic forces and corticotomy. hPDLCs were cultured in vitro and subjected to different mechanical forces. The expression of interleukin (IL)-6 and transforming growth factor (TGF)-β in the supernatant and the mRNA levels of hypoxia inducible factor (HIF)-1α, Notch1, and TGF-β in hPDLCs were investigated. Supernatants were collected and co-cultured with activated CD4+T cells, and the differentiation of Th17/Treg cells was analyzed by flow cytometry. We also established an animal model of tooth movement with or without corticotomy. The tooth movement distance, alveolar bone height, and root resorption were analyzed using micro-computed tomography. Expression of interleukin (IL)-17A, forkhead Box P3 (Foxp3), and IL-6 were analyzed using immunohistochemistry, while osteoclasts were evaluated by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of IL-17A, IL-6, Foxp3, IL-10, HIF-1α, notch1, and C-X-C motif chemokine ligand 12 (CXCL12) in alveolar bone and gingiva were investigated. Heavy force repressed cell viability and increased the mortality rate of hPDLCs; it also improved the expression of IL-6, declined the expression of TGF-β, and promoted the mRNA expression level of HIF-1α. The expression of TGF-β and Notch1 mRNA decreased and then increased. The supernatant of hPDLCs under heavy force promotes the polarization of Th17 cells. The heavy force caused root resorption and decreased alveolar bone height and increased the positive area of IL-17A immunohistochemical staining and the expression of IL-17A, IL-6, HIF-1α, and Notch1 mRNA. Corticotomy accelerated tooth movement, increased the proportion of Foxp3-positive cells, and up-regulated the expression of Foxp3, IL-10, and CXCL12 mRNA. During orthodontic tooth movement, the heavy force causes root resorption and inflammatory bone destruction, which could be associated with increased expression of Th17 cells and IL-6. Corticotomy can accelerate tooth movement without causing root resorption and periodontal bone loss, which may be related to the increased expression of Treg cells. Altogether, this report provides a new perspective on the prevention of inflammatory injury via the regulation of Th17/Treg cells in orthodontics.

Yeast cell wall upregulated cell-mediated immune responses to Newcastle disease virus vaccine

A recent study has suggested that yeast cell wall product (YP) enhanced serum hemagglutination inhibition (HI) titers and intestinal sIgA responses in chickens immunized with Newcastle disease virus (NDV) vaccine. In the present study, the cell-mediated immune responses elicited by NDV and YP were investigated in commercial broilers. Broilers were fed 0 or 0.1% YP and immunized with a live NDV vaccine via an intraocular-and-intranasal route at 14 and 28 days old. After that, blood samples were collected for determination of HI titer, cytokine content, and blood analysis. Eight chickens were randomly selected from each group and sacrificed. Lymphocytes were harvested from the spleens for lymphocyte proliferation and flow cytometry analysis. Total RNA was extracted from spleen and jejunum for RT-qPCR analysis. The results showed that YP significantly increased serum concentration of IL-4, IL-6, IFN-γ, TNF-β, as well as promoted lymphocytes proliferation in broilers immunized with NDV vaccine. The enhanced cell-mediated immunity is correlated with the upregulated mRNA expression of TGF-β, IL-6, TLR5, GATA-3, and T-bet in the spleen and upregulated mRNA expression of CCR-9, J-chain, pIgR, and TLR3 in the jejunum of chickens. It is noteworthy that no significant side effect was observed after the administration of YP. Therefore, YP could be safely used as potential immunopotentiator assisting NDV vaccine for chickens.

Ameliorative effects of pirfenidone in chronic kidney disease

Renal fibrosis is the hallmark of advanced chronic kidney disease (CKD), which is characterized by excessive accumulation of extracellular matrix (ECM) proteins and plays a central role in the pathogenesis and progression of CKD to end-stage renal disease (ESRD). The molecular and cellular substances of kidney fibrosis include growth factors, such as fibroblast growth factor (FGF), transforming growth factor beta (TGF-β) and platelet-derived growth factor (PDGF), alongside cytokines (like interleukin-1b) and metalloproteinases. Therefore, these factors can be evaluated as possible targets for anti-fibrotic agents. Among the mediators of fibrosis, TGF-β is the dominant facilitator of renal fibrosis that induces ECM construction and accumulation. Numerous studies have focused on the inhibition of TGF-β and its downstream targets for the treatment of renal disease. Abolition of TGF-β mRNA expression was found to be the mechanism of anti-fibrotic drug, pirfenidone, in the heart and kidneys of diabetic rats. Various investigations have shown the impact of pirfenidone in diminishing kidney fibrosis, with studies containing patients diagnosed with subtotal nephrectomy, diabetic kidney disease and unilateral ureteral obstruction (UUO), administered drugs such as cyclosporine, tacrolimus, doxorubicin and vanadate. Several therapeutic drugs for fibrosis reduce only one of the oxidative, inflammatory or profibrogenic markers, while pirfenidone targets all three of these markers and therefore, seems to be a particularly valuable drug.

Steamed Panax notoginseng attenuates renal anemia in an adenine-induced mouse model of chronic kidney disease

Ethnopharmacological relevancePanax notoginseng (PN) (Burk.) F. H. Chen is a medicinal herb used to treat blood disorders since ancient times, of which the steamed form exhibits the anti-anemia effect and acts with a “blood-tonifying” function according to the traditional use. However, its pharmacological effect and mechanism on alleviating renal anemia (RA) are still unclear. Aims of the studyThe study aims to investigate the effect of steamed Panax notoginseng (SPN) to attenuate RA and its underlying mechanism based on the model of adenine-induced RA mice. Materials and methodsSeventy mice were randomly divided into seven groups of ten: the control group, model group, the erythropoietin (EPO) group, the Fufang E'jiao Jiang (FEJ) group, the high-dose steamed PN (H-SPN) group, the middle-dose steamed PN (M-SPN) group, and the low-dose steamed PN (L-SPN) group. The adenine induction RA model was applied to assess the “blood enriching” function of SPN. The blood routine indexes, erythrocyte fragility, pathologic morphology of kidney tissue and the expression levels of related cytokines and proteins in the mice were detected after 3-week administration with SPN and positive drugs. ResultsOur study provided evidences that SPN could ameliorate RA. Compared with the control group, SPN could attenuate RA by significantly increasing the numbers of peripheral blood cells (p < 0.01), improving the erythrocyte fragility (p < 0.01), and restoring the expression of EPO mRNA in the kidneys and EPO receptor mRNA in bone marrow nucleated cells. The expression of TGF-β1 mRNA was declined and the expression of HGF mRNA was significantly increased in a dose-dependent way after the treatment of SPN. Additionally, the expression of Bcl-2 and Bcl-2/Bax ratio in the kidneys were significantly increased. In contrast, there was a highly significant decrease in the expression of Bax (p < 0.01), following SPN treatment. ConclusionSPN could alleviate RA by promoting the overall hematopoiesis and inhibiting the progress of renal injury in mice.

The Three Main SCFAs Inhibit the Inflammatory Response of A549 Cells Induced by &amp;lt;i&amp;gt;Acinetobacter baumannii&amp;lt;/i&amp;gt;

Objective: The objective is to explore the mechanism of inhibitory effect of three main SCFAs (acetate, propionate and butyrate) on inflammatory response of A549 cells. Methods: Human lung adenocarcinoma cells (A549 cells) were cultured, and were divided into normal control group (NC group), A. baumannii infection group (A. baumannii group), NF-κB inhibitor group (JSH group), A. baumannii infection + sodium acetate group (NaAc group), A. baumannii infection + sodium propionate group (NaPc group) and A. baumannii infection + sodium butyrate group (NaB group). Real-time quantitative PCR was used to detect the mRNA expression of NLRP3, Caspase-1, IL-1β, IL-6, and TGF-β in A549 cells. Western blotting assay was used to determine the expression of autophagy and “pyroptosis” related proteins of NRLP3, cleaved-Caspase-1 (P20), GSDMD (P30), LC-3 and Beclin-1. At the same time, the expression of NF-κB p65 protein in nucleus and cytoplasm of A549 cells was detected. The level of reactive oxygen species in A549 cells was detected by flow cytometry. Results: Compared with A. baumannii group, the mRNA expression of NLRP3, IL-1β and IL-6 in NaAc group, NaPc group and NaB group decreased significantly, the mRNA expression of Caspase-1 in NaPc group and NaB group decreased significantly, only the mRNA expression of TGF-β in NaB group increased significantly; LC3-II expression increased significantly in NaPc group and NaB group, only Beclin-1 expression increased and GSDMD (p30) expression decreased significantly in NaB group. All three kinds of SCFAs could significantly inhibit the expression of cleaved-Caspase-1 (p20) after A. baumannii infection, but there was no significant change in the protein expression of NLRP3. Compared with NC group, the production of reactive oxygen species in A. baumannii group increased significantly at 3 h after A. baumannii infection. Compared with A. baumannii group, NaB could significantly suppress the production of reactive oxygen species induced by A. baumannii. Compared with A. baumannii group, the expression of NF-κB p65 in nucleus was significantly decreased and the expression of NF-κB p65 in cytoplasm was significantly increased after 24 h pre-incubation with NaB, NaPc and NaAc, respectively. Conclusion: A. baumannii can induce inflammatory injury of pulmonary epithelial cells, and the three major SCFAs can inhibit the activation of NLRP3 inflammasome and the release of pro-inflammatory factors through NF-κB/ROS/NLRP3 pathway, which provides a new way for clinical prevention of severe inflammatory injury caused by A. baumannii infection.

Hyperthermia-induced stellate cell deactivation to enhance dual chemo and pH-responsive photothermal therapy for pancreatic cancers.

For pancreatic ductal adenocarcinoma (PDAC) treatment, the deactivation of pancreatic stellate cells (PSCs) by blocking the transforming growth factor β (TGF-β) pathway is a promising strategy to inhibit stroma, enhance drug penetration, and greatly amplify chemotherapeutic efficacy. It is known that photothermal therapy (PTT) locally depletes stroma and enhances permeability but whether and how PTT reacts in the molecular pathway to induce PSC deactivation in PDAC has rarely been investigated so far. Herein, C-G NPs are synthesized by loading both acid-responsive photothermal molecules and gemcitabine for investigating both the combinatory chemophotothermal therapy and the interaction between the PTT and TGF-β pathway in PDAC. Notably, C-G NPs exhibit tumoral acidic pH-activated PTT and succeeded in deactivating PSCs and suppressing the expression level for both TGF-β and collagen fiber. Furthermore, hyperthermia remodels the tumoral extracellular matrix, significantly improves NP penetration, and boosts the ultimate synergistic chemophotothermal therapeutic efficacy. Importantly, the molecular biology study reveals that hyperthermia leads to the decrease in the mRNA expression of TGF-β1, SMAD2, SMAD3, α-SMA, and Collagen I in the tumor tissue, which is the key to suppress tumor progression. This research demonstrates that combinatory chemophotothermal therapy holds great promise for PDAC treatment.

Expression and possible role of Smad3 in postnecrotizing enterocolitis stricture.

To investigate the expression of Smad3 (mothers against decapentaplegic homolog 3) protein in postnecrotizing enterocolitis stricture and its possible mechanism of action. We used immunohistochemistry to detect the expression characteristics of Smad3 and nuclear factor kappa B (NF-κB) proteins in human postnecrotizing enterocolitis stricture. We cultured IEC-6 (crypt epithelial cells of rat small intestine) in vitro and inhibited the expression of Smad3 using siRNA technique. Quantitative PCR, western blotting, and ELISA were used to detect the changes in transforming growth factor-β1 (TGF-β1), NF-κB, tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and zonula occludens-1 (ZO-1) messenger RNA (mRNA) and protein expressions in IEC-6 cells. CCK8 kit and Transwell cellular migration were used to detect cell proliferation and migration. Changes in epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) in IEC-6 cells were detected by immunofluorescence technique. The results showed that Smad3 protein and NF-κB protein were overexpressed in narrow intestinal tissues and that Smad3 protein expression was positively correlated with NF-κB protein expression. After inhibiting the expression of Smad3 in IEC-6 cells, the mRNA expressions of NF-κB, TGF-β1, ZO-1, and VEGF decreased, whereas the mRNA expression of TNF-α did not significantly change. TGF-β1, NF-κB, and TNF-α protein expressions in IEC-6 cells decreased, whereas ZO-1 and intracellular VEGF protein expressions increased. IEC-6 cell proliferation and migration capacity decreased. There was no significant change in protein expression levels of EMT markers E-cadherin and vimentin and also extracellular VEGF protein expression. We suspect that the high expression of Smad3 protein in postnecrotizing enterocolitis stricture may promote the occurrence and development of secondary intestinal stenosis. The mechanism may be related to the regulation of TGF-β1, NF-κB, TNF-α, ZO-1, and VEGF mRNA and protein expression. This may also be related to the ability of Smad3 to promote epithelial cell proliferation and migration.

Exploration of the Effects of TGF-β Pathway-Based Pituitary Tumor of Rats on GH3 Cell Line after Intervention with Different Concentrations of TGZ.

The effect of the TGF-β pathway-based pituitary tumor of rats on the GH3 cell line after intervention with different concentrations of troglitazone (TGZ) is explored. The CH3 cell line of 24 clean male SD rats with pituitary adenoma is selected. The cells are divided into a blank contrast set and an experimental set. The experimental set is divided into different TGZ concentration sets, including 1 × 10−3 TGZ set, 1 × 10−4 TGZ set, and 1 × 10−5 TGZ set. The cell proliferation is detected by the CCK-8 method, the protein expressions of CD147, TGF-β1, and MMP-9 are detected by the western blot method, and the relative mRNA expressions of CD147, TGF-β1, and MMP-9 are detected by the qRT-PCR method. The expression levels of CD147, TGF-β1, and MMP-9 in CH3 cells of pituitary adenoma rats are notoriously lower, while the expression of CD147, TGF-31, and MMP-9 could be reduced by TGZ acting on the GH3 cell line. The specific mechanism of action of this effect on the invasive ability of GH3 cell lines is multifaceted, suggesting that peroxisome proliferator activator-receptor (PPAR-γ) agonists have good clinical application prospects in tumor therapy and can provide new targets and approaches for tumor drug therapy.

Evaluate the effect of licorice on anti-liver fibrosis: a systematic review and meta-analysis

To investigate the effect of licorice on anti-liver fibrosis actions, we present systematic review and meta-analysis via systematic literature between 2010 and 2020 from the electronic databases, PubMed, Cochrane Library, Embase, ISI. A software program (Endnote X8) has been utilized for managing the electronic titles. Searches were performed with mesh terms. Mean differences between two groups (Glycyrrhizic acid and control group) with 95% confidence interval (CI), fixed effect model and Inverse-variance method were calculated. Random effects were used to deal with potential heterogeneity and I2 showed heterogeneity. The Meta analysis and forest plots have been evaluated with the Stata V16. A total of 184 potentially relevant titles and abstracts were found during the electronic and manual search. Finally, a total of four publications fulfilled the inclusion criteria required for this systematic review. TGF-β1 mRNA expression significantly decreased following Glycyrrhizic acid relative to the control group and Glycyrrhizic acid significantly decreased expression of TGF-β1 mRNA, which was about 2.90 times that in the control group. Mean difference between Glycyrrhizic acid and control group was -18.12 U/L (MD, -18.12 U/L 95% CI 30.02 U/L, -6.22 U/L. P = 0.00). Glycyrrhizic acid reduced TGF-β1, Smads mRNAs, hypdroxyproline, alanine aminotransferase.

Effects of Signaling Activation of Bone Morphogenetic Protein 2 on the Differentiation and Infiltration of Regulatory T Lymphocytes in Tumors

Objective To determine whether the signaling activation of bone morphogenetic protein 2(BMP2)can induce myeloid-derived suppressor cells(MDSC)to secret transforming growth factor β(TGF-β),further enhancing the differentiation and infiltration of regulatory T lymphocytes(Treg)into tumor tissue. Methods The BMP2-induced mRNA and protein expression of TGF-β in MDSC was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay(ELISA),respectively.The effect of BMP2-induced TGF-β secretion by MDSC on Treg differentiation was then determined by flow cytometry.Finally,we implanted the recombined human bone morphogenetic protein 2(rhBMP2)collagen gels into tumor-burdened mice to examine the role of BMP2 in Treg differentiation via MDSC-secreted TGF-β in vivo.The protein levels of TGF-β in peripheral blood and tumor tissue were detected by ELISA,and the infiltration of Treg cells in tumor tissue was detected by immunofluorescence assay. Results BMP2 up-regulated the mRNA and protein levels of TGF-β in MDSC in vitro.TGF-β secreted by the MDSC exposed to BMP2 treatment could induce the differentiation of Treg cells in vitro.Local implantation of rhBMP2 could increase the level of TGF-β protein in peripheral blood and tumor tissue of mice,further enhancing the infiltration of Treg cells into tumors. Conclusion BMP2 signaling activation can induce the differentiation of Treg cells by promoting the secretion of TGF-β in MDSC,and subsequently promote the infiltration of Treg cells into tumors.

Improved bladder contractility after transplantation of human mesenchymal stem cells overexpressing hepatocyte growth factor into underactive bladder from bladder outlet obstruction models of rats.

IntroductionAn underactive bladder can lead to difficulty in voiding that causes incomplete emptying of the bladder, suggesting the need for a new strategy to increase bladder contractility in such patients. This study was performed to investigate whether human mesenchymal stem cells (hMSCs) were capable of restoring bladder contractility in rats with underactive bladder due to bladder outlet obstruction (BOO) and enhancing their effects by overexpressing hepatocyte growth factor (HGF) in hMSCs.Materials and methodsThe hMSCs were transplanted into the bladder wall of rats. Fifty female Sprague-Dawley rats at six weeks of age were divided into five groups: group 1: control; group 2: sham intervention; group 3: eight-week BOO; group 4: BOO rats transplanted with hMSCs; and group 5: BOO rats transplanted with hMSCs overexpressing HGF. Two weeks after the onset of BOO in groups 4 and 5, hMSCs were injected into the bladder wall. Cystometry evaluation was followed by Masson’s trichrome staining of bladder tissues. Realtime PCR and immunohistochemical staining were performed to determine for hypoxia, apoptosis, and angiogenesis.ResultsCollagen deposition of bladder increased in BOO but decreased after transplantation of hMSCs. The increased inter-contraction interval and residual urine volume after BOO was reversed after hMSCs transplantation. The decreased maximal voiding pressure after BOO was restored by hMSCs treatment. The mRNA expression of bladder collagen1 and TGF-β1 increased in BOO but decreased after hMSCs transplantation. The decrease in vWF-positive cells in the bladder following BOO was increased after hMSCs transplantation. Caspase 3 and TUNEL-positive apoptosis of bladder cells increased in BOO but decreased after transplantation of hMSCs. These effects were enhanced by overexpressing HGF in hMSCs.ConclusionTransplantation of hMSCs into bladder wall increased the number of micro-vessels, decreased collagen deposition and apoptosis of detrusor muscle, and improved bladder underactivity. The effects were enhanced by overexpressing HGF in hMSCs. Our findings suggest that the restoration of underactive bladder using hMSCs may be used to rectify micturition disorders in patients following resolution of BOO. Further studies are needed before hMSCs can be used in clinical applications.

Transplantation of IL-1β siRNA-modified bone marrow mesenchymal stem cells ameliorates type II collagen-induced rheumatoid arthritis in rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes erosion of articular cartilage and bone and has adverse effects on both patients and livestock animals. The aim of the present study was to investigate the role of interleukin-1β (IL-1β) in the pathogenesis of RA, and to further determine whether injection of IL-1β small interfering RNA (siRNA) or transplantation of IL-1β siRNA + bone marrow mesenchymal stem cells (BMSCs) can ameliorate RA in rats. A collagen-induced arthritis (CIA) rat model was established by injecting type II collagen for 4 weeks. Next, CIA rats were randomly divided into three groups and injected or transplanted with PBS, IL-1β siRNA and IL-1β siRNA + BMSCs for another 4 weeks. The CIA rat model was successfully established, as demonstrated by the higher toe swelling value, thymus and spleen/body weight, immobility time and serum IL-1β concentration, as well as lower body weight, climbing time and mRNA expression of programmed death-1 (PD-1), transforming growth factor-β1 (TGF-β1) and forkhead box protein 3 (Foxp3) in the spleen, compared with control rats. Furthermore, histopathology results demonstrated that joint swelling and redness were observed in the knee joints of CIA rats. H&E results revealed that CIA rats presented erosive destruction of the bone and ulceration of the articular cartilage. In addition, in vitro results demonstrated that IL-1β expression was successfully silenced after IL-1β siRNA transfection in lipopolysaccharide-stimulated BMSCs. When compared with the results of PBS rats, both IL-1β siRNA injection and IL-1β siRNA + BMSC transplantation significantly increased the body weight, climbing time and mRNA expression of PD-1, TGF-β1 and Foxp3 in the spleen, while significantly reduced the immobility time and serum IL-1β concentration. In addition, when compared with that of IL-1β siRNA injection, IL-1β siRNA + BMSC transplantation exhibited markedly higher therapeutic efficacy against CIA. These results demonstrated that higher IL-1β contributed to the pathogenesis of CIA, and that IL-1β siRNA injection ameliorated CIA, while its combination with BMSCs exerted synergistic effects, which may be beneficial against RA.

Metabolomics and pharmacodynamic analysis reveal the therapeutic role of Prunella vulgaris oil on intrauterine adhesion rats

Metabolomics is applied to explore the curative effect of complex systems, such as Chinese medicine. Intrauterine adhesion (IUA) harms the reproductive system and affects fertility, and hence is a significant public health concern. Prunella vulgaris oil (PVO) protects the reproductive system and exerts anti-inflammatory effects, but its effect on IUA and the underlying mechanism is unclear. In this study, we established a serum metabolomics method based on GC-TOF-MS to evaluate the mechanism of PVO in the IUA rat model established by mechanical injury and infection. Animal experiments showed that PVO improves the inflammatory response in the uterus of IUA model rats and reduces the content of inflammatory factors to improve the microenvironment of the reproductive system. It also regulates the expression of TGF-β1 and Smad-related mRNA and protein to inhibit fibrosis. Metabolomics indicated a significant abnormality in serum metabolism in IUA rats, and a total of 51 differential markers were screened and identified. After PVO treatment, these metabolic abnormalities improved significantly. The metabolic pathway analysis revealed that PVO affects glyoxylate and dicarboxylate metabolism, and β-alanine metabolism pathways. This study showed that PVO significantly improves inflammation and fibrosis in IUA rats combined with the pharmacological results. The primary mechanism is related to regulating the metabolism of amino acids and their derivatives to balance the associated disorders and control energy metabolism.

MicroRNA miR-30a inhibits cisplatin resistance in ovarian cancer cells through autophagy

ABSTRACT We study whether microRNA miR-30a inhibits the autophagy through transforming growth factor (TGF)-β/Smad4 to generate cisplatin (DDP) resistance in ovarian cancer cells. The expression of miR-30a, Smad4, and TGF-β was detected in the serum of ovarian cancer patients and DDP-resistant cell lines (A2780) by quantitative real-time polymerase chain reaction (qRT-PCR). Computational search and western blotting were used to demonstrate the downstream target of miR-30a in ovarian cancer cells. Cell viability was measured with CCK8 assay. Apoptosis and autophagy of ovarian cancer cells were analyzed by flow cytometry and transmission electron microscopy, and the expressions of Beclin1 and LC3II protein were detected by western blotting. Expression of miR-30a was significantly decreased, while expressions of TGF-β and Smad4 mRNA were increased in serum of ovarian cancer patients after DDP chemotherapy as well as in DDP-resistant cells. Activation of autophagy contributed to DDP-resistance cells. Moreover, Bioinformatics software predicted Smad4 to be a target of miR-30a. Overexpression of miR-30a decreased the expression of Smad4 and TGF-β. Additionally, miR-30a-overexpressing inhibited DDP-induce autophagy and promoted DDP-resistant cell apoptosis. In conclusion, miR-30a mediates DDP resistance in ovarian cancer by inhibiting autophagy via the TGF-β/Smad4 pathway.

Influence of leptin and compression in GAS-6 mediated homeostasis of periodontal ligament cell.

Growth arrest-specific protein 6 (GAS-6) regulates immunomodulatory and inflammatory mechanisms in periodontium and may participate in obesity predisposition. This study aimed to determine whether GAS-6 is associated with the homeostasis of periodontal ligament (SV-PDL) cells in the presence of adipokines or compressive forces. The SV-PDL cell line was used. Western blots were employed for TAM receptors detection. Cells were stimulated using different concentrations of GAS-6. The migration, viability, and proliferation were measured by a standard scratch test, MTS assay, and immunofluorescent staining. The mRNA expression was analyzed by RT-PCR. Release of TGF-β1, GAS-6, and Axl were verified by ELISA. Western blot shows that TAM receptors are expressed in SV-PDL cells. GAS-6 has a promoting effect on cell migration and proliferation. RT-PCR analysis showed that GAS-6 induces Collagen-1, Collagen-3, Periostin, and TGF-β1 mRNA expression whereas it reduces Caspase-3, Caspase-8, Caspase-9, and IL-6 mRNA expression. Further, secreted GAS-6 in SV-PDL is reduced in response to both compressive forces and leptin and upregulated by IL-6. Additionally, ADAM-10 inhibition reduces GAS-6 and Axl release on SV-PDL cells. TAM receptors especially Axl are identified as the receptors of GAS-6. GAS-6/TAM interactions contribute to periodontal ligament cells homeostasis. Leptin inhibits the GAS-6 release independently of ADAM-10 metalloprotease.

The effect of 4-Week rehabilitation by aerobic exercise on hippocampus BDNF and TGF-β1 gene expressions in Aβ 1–42 -induced rat model of Alzheimer’s disease

Alzheimer’s disease (AD) is a type of brain dysfunction featuring a gradual loss in memory. This study aimed to determine the effect of 4 weeks of aerobic rehabilitation exercise (RhExe) on the genes expression of BDNF and TGF-β1 in the hippocampus tissue of rats with the AD induced by injection of amyloid-beta (Aβ1-42). Twenty-one male Wistar rats were randomly divided into 3 groups: Aβ injection (n = 7), Aβ + exercise (n = 7) and control (n = 7). AD was induced by a single dose of Aβ injection into the hippocampus of rats. Three days after surgery, the Aβ + exercise group experienced four weeks of the RhExe (5 days/week). Forty-eight hours after the last training session, the animals underwent the Morris water maze test. The animals were sacrificed 24 h after the test, and hippocampal tissue was split. The mRNA expression of BDNF, TGF-β1, and TGF-β1 II receptors was measured. The TGF-β1 and TGF-β1 II receptor genes expression of Aβ + exercise group were significantly higher than the Aβ injection group (P ≤ 0.001). BDNF gene expression in the hippocampus of the Aβ + exercise group was significantly higher than the Aβ injection group (P ≤ 0.001). Spatial memory was significantly higher in the Aβ + exercise group than in the Aβ injection group (p ≤ 0.01). It seems that aerobic exercise can counteract the harmful effects of Aβ through the BDNF and TGF-β1molecular signaling pathways.

PM2.5 Induces Airway Remodeling in Chronic Obstructive Pulmonary Diseases via the Wnt5a/β-Catenin Pathway.

BackgroundFine-particulate matter ≤2.5 μm in diameter (PM2.5)-associated airway remodeling has recently been recognized as a central feature of COPD. Activation of the Wnt/β-catenin pathway is closely related to the occurrence of airway remodeling. Accordingly, the goal of this study was to determine whether the Wnt5a/β-Catenin pathway is involved in PM2.5-induced smooth muscle proliferation in vivo and in vitro, which promotes the development of airway remodeling in subjects with COPD.MethodsThe effect of Wnt5a on β-Catenin-mediated airway remodeling was assessed using an in vivo model of PM2.5-induced COPD and PM2.5-exposed human bronchial smooth muscle cells (HBSMCs) in vitro. Small animal spirometry was used to measure lung function in mice. H&E staining and immunohistochemistry were performed to inspect emphysema and airway remodeling indices. Real-time PCR was used to detect Wnt5a, β-Catenin, TGF-β1, CyclinD1 and c-myc mRNA expression. The CCK8 assay was performed to detect cellular activity. Western blotting was performed to assess PCNA, α-SMA, Wnt5a, β-Catenin, PDGFRβ and TenascinC protein expression. β-Catenin expression was detected using cellular immunofluorescence.ResultsExposure to PM2.5 led to emphysema, airway wall thickening, an increased smooth muscle layer thickness, decreased lung function and increased expression of the Wnt5a, β-Catenin, PDGFRβ and Tenascin C proteins in the mouse lung tissue. BOX5 (a Wnt5a antagonist) alleviated these PM2.5-induced outcomes in mice. Moreover, PM2.5 induced the expression of the Wnt5a, β-Catenin, TGF-β1, CyclinD1 and c-myc mRNAs in HBSMCs. BOX5 also inhibited the PM2.5-induced increases in PCNA, α-SMA, Wnt5a, β-Catenin, PDGFRβ and Tenascin C protein expression in HBSMCs.ConclusionOur findings suggest that PM2.5 exposure induces HBSMC proliferation, contributing to airway remodeling via the Wnt5a/β-Catenin signaling pathway in vivo and in vitro, which might be a target for COPD treatment.