Protein Expression Levels Of TGF-β1 Research Articles

Marein Ameliorates Myocardial Fibrosis by Inhibiting HIF-1α and TGF-β1/Smad2/3 Signaling Pathway in Isoproterenol-stimulated Mice and TGF-β1-stimulated Cardiac Fibroblasts.

Myocardial fibrosis significantly contributes to the pathogenesis and progression of heart failure. We probe into the impact of marein, a key bioactive compound in functional food Coreopsis tinctoria, on isoproterenol-stimulated myocardial fibrotic mice and transforming growth factor β1 (TGF-β1)-stimulated cardiac fibroblasts (CFs). Isoproterenol was administered to the experimental mice via subcutaneous injection, and simultaneous administration of marein (25-100 mg/kg) was performed via oral gavage. CFs were stimulated with TGF- β1 to trigger differentiation and collagen synthesis, followed by treatment with marein at concentrations of 5-20 μM. Treatment with marein in mice and CFs resulted in a significant reduction in the protein expression levels of α-smooth muscle actin, collagen type I, and collagen type III. Additionally, marein treatment decreased the protein expression levels of TGF-β1, hypoxia-inducible factor-1α (HIF-1α), p-Smad2/3, and Smad2/3. Notably, molecular docking analysis revealed that marein directly targets HIF-1α. Marein might exert a protective function in isoproterenol-stimulated myocardial fibrotic mice and TGF-β1-stimulated CFs, which might result from the reduction of TGF-β1 induced HIF-1α expression, then inhibiting p-Smad2/3 and Smad2/3 expressions.

Effect of mycophenolate mofetil alleviates carbon tetrachloride-induced liver fibrosis in mice.

Hepatic fibrosis is a serious pathological consequence of chronic liver disease. Mycophenolate mofetil (MMF) is a commonly used immunosuppressant after organ transplant. However, the relationship between MMF and hepatic fibrosis remains unclear. This study aims to explore the effect of MMF on hepatic fibrosis in mice and the potential mechanism. A total of 24 mice (male, 8-week old, C57BL/6) were randomly divided into a control group, a MMF group, a carbon tetrachloride (CCl4) group and a CCl4+MMF group (n=6 in each group). After the mice were sacrificed, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected. The liver tissues were taken up for Masson staining and collagen I (COL1) immunohistochemistry. The levels of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were detected by Western blotting. Finally, the levels of mRNA for TGF-β1, α-SMA, and COL1 were detected using real-time PCR. Compared with the CCl4 group, the ALT and AST levels were lower (both P<0.05), the degree of liver fibrosis was alleviated, and the deposition of COL1 in the liver was significantly decreased (P<0.01) in the CCl4+MMF group. Compared with the CCl4 group, the protein expression levels of TGF-β1 and α-SMA were significantly decreased (both P<0.05) and the relative expression levels of TGF-β1, α-SMA and COL1 mRNA in the liver were significantly decreased (all P<0.05) in the CCl4+MMF. MMF could reduce CCl4-induced hepatic fibrosis, which might be related to the inhibition of TGF-β1. This study is expected to provide a target for the treatment of hepatic fibrosis.

The effect of extracorporeal shock wave on joint capsule fibrosis in rats with knee extension contracture: a preliminary study

ABSTRACT The purpose of this study was to observe the therapeutic effect of extracorporeal shock wave (ESW) on extensional joint contracture of knee joint in rats and its mechanism on articular capsule fibrosis. Thirty-two SD rats were randomly divided into blank control, immobilization, natural recovery, and ESW intervention groups. Except for the control group, the left knee joints of other rats were fixed with external fixation brace for 4 weeks when they were fully extended to form joint contracture. The effect of intervention was assessed by evaluating joint contracture, total cell count and collagen deposition in joint capsule, and protein expression levels of TGF-β1, p-Smad2/3, Smad2/3, p-JNK, JNK, I and III collagen in joint capsule. ESW can effectively reduce arthrogenic contracture, improve the histopathological changes of anterior joint capsule, inhibit the high expression of target protein and the excessive activation of TGF-β1/Smad2/3/JNK signal pathway. Inhibition of excessive activation of TGF-β1/Smad2/3/JNK pathway may be one of the potential molecular mechanisms by which extracorporeal shock wave can play a role.

A potent and selective CXCR2 antagonist improves neuroimmune dysregulation through the inhibition of NF-κB and notch inflammatory signaling in the BTBR mouse model of autism.

Autism comprises a broad range of neurodevelopmental disorders characterized by social communication deficits and repetitive and stereotyped behaviors. Chemokine receptor CXCR2 is expressed on neurons and is upregulated in neurological disorders. BTBR T+ Itpr3tf/J (BTBR) mice, a model for autism that shows the core features of ASD. Here, we studied the anti-inflammatory effect of a potent and selective CXCR2 antagonist SB332235 in the BTBR mice. The CXCR2 antagonist represents a promising therapeutic agent for several neuroinflammatory disorders. In this study, we investigated the effects of SB332235 administration on NF-κB-, Notch-1-, Notch-3-, GM-CSF-, MCP-1-, IL-6-, and IL-2- and TGF-β1-expressing CD40+ cells in BTBR and C57BL/6 (C57) mice in the spleen cells by flow cytometry. We further assessed the effect of SB332235 treatment on NF-κB, Notch-1, GM-CSF, MCP-1, IL-6, and IL-2 mRNA expression levels in the brain tissue by RT-PCR. We also explored the effect of SB332235 administration on NF-κB, GM-CSF, IL-6, and TGF-β1 protein expression levels in the brain tissue by western blotting. The SB332235-treated BTBR mice significantly decreases in CD40+NF-κB+, CD40+Notch-1+, CD40+Notch-3+, CD40+GM-CSF+, CD40+MCP-1+, CD40+IL-6+, and CD40+IL-2+, and increases in CD40+TGF-β1+ in the spleen cells. Our results further demonstrated that BTBR mice treated with SB332235 effectively decreased NF-κB, Notch-1, GM-CSF, MCP-1, IL-6, and IL-2, increasing TGF-β1 mRNA and protein expression levels in the brain tissue. In conclusion, these results indicate that SB332235 elicits an anti-inflammatory response by downregulating the inflammatory mediators and NF-κB/Notch inflammatory signaling in BTBR mice. This could represent a promising novel therapeutic target for autism treatment.

Regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts

Objective: To investigate the regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts (HSFbs). Methods: The experimental research methods were used. The 4th-6th passage of HSFbs from human skin were used for the following experiments. HSFbs were co-cultured with sodium ferulate at final mass concentrations of 1, 1×10-1, 1×10-2, 1×10-3, 1×10-4, 1×10-5, and 1×10-6 mg/mL for 48 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and linear regression was used to analyze the half lethal concentration (LC50) of sodium ferulate (n=6). HSFbs were co-cultured with sodium ferulate at final mass concentrations of 0.1, 0.2, 0.3, and 0.4 mg/mL for 24, 48, 72, and 96 hours, and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and the cell proliferation inhibition rate was calculated (n=3). According to the random number table, the cells were divided into 0.300 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, 0.003 mg/mL sodium ferulate group treated with sodium ferulate at corresponding final mass concentrations, and negative control group without any treatment. After 72 hours of culture, the cell absorbance values were determined by methyl thiazolyl tetrazolium method (n=5), the microscopic morphology of cells was observed by transmission electron microscope (n=3), the cell apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and the apoptosis index was calculated (n=4), the protein expressions of B lymphocystoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteine aspartic acid specific protease-3 (caspase-3) were determined by immunohistochemistry (n=4), and the protein expressions of transformed growth factor β1 (TGF-β1), phosphorylated Smad2/3, phosphorylated Smad4, and phosphorylated Smad7 were detected by Western blotting (n=4). Data were statistically analyzed with one-way analysis of variance and Dunnett test. Results: The LC50 of sodium ferulate was 0.307 5 mg/mL. After being cultured for 24-96 hours, the cell proliferation inhibition rates of cells treated with sodium ferulate at four different mass concentrations tended to increase at first but decrease later, which reached the highest after 72 hours of culture, so 72 hours was chosen as the processing time for the subsequent experiments. After 72 hours of culture, the cell absorbance values in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were 0.57±0.06, 0.53±0.04, 0.45±0.05, respectively, which were significantly lower than 0.69±0.06 in negative control group (P<0.01). After 72 hours of culture, compared with those in negative control group, the cells in the three groups treated with sodium ferulate showed varying degrees of nuclear pyknosis, fracture, or lysis, and chromatin loss. In the cytoplasm, mitochondria were swollen, the rough endoplasmic reticulum was expanded, and local vacuolation gradually appeared. After 72 hours of culture, compared with that in negative control group, the apoptosis indexes of cells were increased significantly in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group (P<0.05 or P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expressions of Bcl-2 of cells in 0.300 mg/mL sodium ferulate group was significantly decreased (P<0.01), the protein expressions of Bax of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.05), and the protein expression of caspase-3 of cells in 0.300 mg/mL sodium ferulate group was significantly increased (P<0.01). After 72 hours of culture, compared with those in negative control group, the protein expression levels of TGF-β1, phosphorylated Smad2/3, and phosphorylated Smad4 of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly decreased (P<0.05 or P<0.01), and the protein expression levels of phosphorylated Smad7 of cells in 0.003 mg/mL sodium ferulate group, 0.030 mg/mL sodium ferulate group, and 0.300 mg/mL sodium ferulate group were significantly increased (P<0.01). Conclusions: Sodium ferulate can inhibit the proliferation of HSFbs of human skin and promote the apoptosis of HSFbs of human skin by blocking the expression of key proteins on the TGF-β/Smad signaling pathway and synergistically activating the mitochon- drial apoptosis pathway.

Wound healing potential of forest honey for increasing TGF-β1 protein expression in palatoplasty: In-vivo and In-silico studies

Background: With negative side effects of a lengthy healing time and high complication rate, cleft palate surgery is employed to treat cleft palate, which is a common congenital anomaly. Honey is believed to help accelerate wound healing post-treatment. However, its effect on the increase in the transforming growth factor (TGF)-β1 expression after palate surgery is unclear. Objective: This study investigates the effect of forest honey on TGF-β1 protein expression levels in the wound healing palate model. Methods: This study evaluates the TGF-β1 protein expression of a palatoplasty wound model using enzyme-linked immunosorbent assay. For this purpose, a punch biopsy model with 30 Sprague-Dawley rats is treated with forest honey (TG), Aloclair Gel (PC), and distilled water (NC) for 3 days. Analysis of the TGF-β1 expression on day 4 is performed by statistical one-way analysis of variance and post hoc least significance difference, with a 0.05 significant P-value. Online website software helped to predict the effect of honey components on the TGF-β1 expression. Results: Protein levels of treatment group (T), negative control (NC), and positive control (PC) exhibit mean levels of 16.13 ± 1.06883 ng/L, 7.36 ± 0.16543 ng/L, and 15.03 ± 0.34221 ng/L, respectively. The differential expression T group exhibits a 2.19-fold change in TGF-β1 relative to the NC group and a 1.07-fold change in the PC group (P-value of 0.01). TGF expression in the PC group increases in comparison to that in the NC group by 2.04-fold (P-value of 0.01). In-silico analysis revealed that genistein promotes macrophage proliferation and activation via the increase in the TGF-β1 expression. Conclusion: In summary, forest honey can boost the TGF protein expression via genistein to increase macrophage proliferation and activation.

Dendrobium Mixture Ameliorates Diabetic Nephropathy in db/db Mice by Regulating the TGF-β1/Smads Signaling Pathway.

Dendrobium mixture (DMix) is an effective treatment for diabetic nephropathy (DN), but the molecular mechanism underlying its action remains unclear. In this study, we investigated whether DMix regulates the transforming growth factor-β1 (TGF-β1)/Smads signal transduction pathway. Twenty-four db/db mice were randomly divided into three groups: the model, DMix, and gliquidone groups, while eight db/m mice were selected as the normal control group. The drug was administered by continuous gavage for 8 weeks. Body weight (BW), kidney weight (KW), kidney index, fasting blood glucose (FBG), blood lipid, 24-hour urinary albumin excretion rate, blood urea nitrogen, and serum creatinine levels were measured. Pathological changes in the renal tissue were observed under a light microscope. Real-time quantitative PCR and immunohistochemical staining were used to detect the mRNA and protein expression levels of TGF-β1 and alpha-smooth muscle actin (α-SMA), respectively, in renal tissues. TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3, and α-SMA expression levels were measured using western blotting. The results showed that DMix significantly reduced the FBG level, BW, KW, and blood lipid level and improved renal function in db/db mice. Histopathology showed that DMix alleviated glomerular mesangial cell proliferation and renal interstitial fibrosis in db/db mice. Additionally, DMix reduced the protein and mRNA expression levels of TGF-β1 and α-SMA and inhibited Smad2 and Smad3 phosphorylation. We conclude that DMix may inhibit renal fibrosis and delay the progression of DN by regulating the TGF-β1/Smads signaling pathway.

Prenatal EGCG exposure-induced heart mass reduction in adult male mice and underlying mechanisms

Epigallocatechin-3-gallate (EGCG), which is a major polyphenol in tea, has an unclear effect on cardiac development. In the present study, mice (C57BL/6) were exposed in utero to EGCG dissolved in drinking water (3 μg/ml) for 16 days. A significant decrease in the heart/body weight ratio was observed in adult males but not in adult females. The protein expression levels of TGF-β1 and its downstream transcription factors SMAD3 and SMAD4 were significantly decreased in male hearts. The PI3K/AKT signaling pathway was inhibited, the expression of proapoptotic proteins, such as BAX, Cleaved Caspase3 and Cleaved Caspase9, was elevated, and the level of antiapoptotic proteins, such as BCL-2, was decreased. A reduced heart/body weight ratio may be associated with the loss of cardiac fibers and an increase in myocardial apoptosis. The cardiac levels of aromatic hydrocarbon receptor and androgen receptor were elevated only in males, which may explain the sexual dimorphism in the effects. The promoter methylation levels of pik3r1, tgf-β, smad4 were elevated, and those of ahr were reduced, explaining the mechanism underlying the cardiac histological alteration caused by prenatal exposure to EGCG. The results suggest that ingestion of EGCG during pregnancy may be a risk factor for cardiac development in offspring.

Drug-containing serum of rhubarb-astragalus capsule inhibits the epithelial-mesenchymal transformation of HK-2 by downregulating TGF-β1/p38MAPK/Smad2/3 pathway

Ethnopharmacological relevanceRheum palmatum L; Astragalus membranaceus (Fisch.), is referred to as ‘Dahuang, Huangqi’ in China. As an important medicinal plant, the rhizome of rhubarb and astragalus is traditionally used in the treatment of kidney diseases associated with renal failure, inflammation and tumors.Aim of the study: This study aimed to investigate the effect of a drug-containing serum of rhubarb-astragalus capsules (composed of rhubarb and astragalus) and to elucidate its mechanism in the epithelial-mesenchymal transformation of renal tubular epithelial cells. Materials and methodsEpithelial-mesenchymal transformation (EMT) of HK-2 cells was induced by TGF-β1, and rhubarb-astragalus and losartan drug-containing serum from rats, as well as SB203580 (a specific inhibitor of p38 MAPK), were used. High-performance liquid chromatography analysis was performed to determine the main components of the drug-containing serum of rhubarb-astragalus from rats. Western blotting and immunofluorescence analysis were used to determine the levels of protein expression, and real-time quantitative PCR analysis was used to detect the levels of gene expression. ResultsThe drug-containing serum of rhubarb-astragalus contained emodin (0.36 μg/ml) and danthraquinone (0.96 μg/ml). Rhubarb-astragalus significantly decreased the protein expression levels of α-SMA, FN, vimentin and N-cadherin in HK-2 cells that were increased by TGF-β1, while it significantly increased the E-cadherin protein expression level that was decreased by TGF-β1. Rhubarb-astragalus also significantly decreased the protein expression levels of TGF-β1 and p38 MAPK and the mRNA expression levels of α-SMA, vimentin, TGF-β1, p38 MAPK, Smad2 and Smad3 in HK-2 cells that were increased by TGF-β1. It is worth noting that SB203580 (a p38 MAPK inhibitor) had similar effects as rhubarb-astragalus in this study. ConclusionThe drug-containing serum of rhubarb-astragalus can inhibit EMT in HK-2 cells by downregulating the TGF-β1/p38 MAPK/Smad2/3 pathway.

The Role of Smad2 in Transforming Growth Factor β1–Induced Hypertrophy of Ligamentum Flavum

Hypertrophy of the ligamentum flavum (LF) contributes to the development of spinal stenosis. Smad proteins can mediate the fibrogenesis activity through the transforming growth factor β1 (TGF-β1) pathway, but which Smad protein plays a more important role in the hypertrophy process of LF is unclear. The LF samples were obtained from 50 patients. After the LF cells (LFCs) were cultured, small interfering ribonucleic acid (siRNA) that target human phosphorylated-Smad2, 3, or 4 (p-Smad2,3,4) genes was transfected into LFCs. Next, proteins from cells were extracted and the protein levels of Smad2, Smad3, and Smad4 were detected by Western blot. The messenger ribonucleic acid level of TGF-β1 was measured by real-time polymerase chain reaction (PCR). Furthermore, an enzyme-linked immunosorbent assay was performed to test the impact of Smad2 downstream of the TGF-β1 signaling pathway. Degeneration of the LF was characterized by an increase in disorganized elastic fibers and fibrotic transformation by extracellular collagen deposition. The gene expression analysis of fibrotic genes in LFCs showed that knockdown of phosphorylated-Smad2 by siRNA significantly reduced the protein expression level of TGF-β1 compared with other groups. The enzyme-linked immunosorbent assay suggested that the protein expression level of Smad2 can influence the downstream events of TGF-β1 signaling pathway in the LFCs. Our findings suggest that Smad2 plays a potential role in the pathologic development of hypertrophy of LF. We also found that Smad2 knockdown by Smad-siRNA can influence the TGF-β1 signaling pathway through decreasing expression of TGF-β1, tumor necrosis factor α, and nuclear factor κb.

Irbesartan ameliorates myocardial fibrosis in diabetic cardiomyopathy rats by inhibiting the TGF&amp;beta;1/Smad2/3 pathway

Myocardial fibrosis (MF) is an important pathological change in diabetic cardiomyopathy. The aim of the present study was to investigate whether irbesartan serves a role in improving MF in a diabetic rat model. Fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured in rats using biochemical methods. Heart weight index (HWI), left ventricular weight index (LVWI), left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were also measured, whilst type I collagen and hydroxyproline content in myocardial tissue was quantified. Western blotting was used to measure the expression of transforming growth factor β1 (TGFβ1), phosphorylated (p)-Smad2/3 and collagen type I α 1 chain (COL1A1) inmyocardial tissues or rat cardiac fibroblast (RCF) cells. Cell proliferation was measured using EdU staining. Procollagen type III N-terminal peptide (PIIINP) content, FBG, TC, TG and LDL-C levels were found to be significantly higher, whilst HDL-C levels were found to be significantly lower in rats in the diabetic group. Those in the diabetic group also exhibited significantly elevated HWI, LVWI, LVEDP, myocardial tissue type I collagen content and hydroxyproline content values, but significantly reduced LVSP. Changes in the aforementioned indicators were reversed after treatment with irbesartan, where the protein expression levels of TGFβ1 and p-Smad2/3 in myocardial tissue were also significantly reduced. In RCF cells, irbesartan significantly reversed high glucose-induced upregulation of TGFβ1 expression, Smad2/3 phosphorylation and COL1A1 expression, as well as reducing cell proliferation and rat type I PICP and PIIINP levels. Application of pirfenidone produced additive effects on reducing the expression levels of the proteins aforementioned when combined with irbesartan. Therefore, the present results demonstrated that irbesartan reduced the activity of the TGFβ1/Smad2/3 pathway and ameliorated diabetic MF by downregulating the expression of TGFβ1.

Effects of maslinic acid on isoproterenol-induced myocardial fibrosis in mice

Objective: To investigate the effect of maslinic acid (MA) on isoproterenol (ISO)-induced myocardial fibrosis in mice. Methods: ISO was used to induce myocardial fibrosis in adult male C57BL/6 mice, and MA was administered for two weeks to detect the effects of MA on cardiac function and fibrosis. Molecular changes of fibrosis markers and signaling pathways were detected by RT-PCR and western blotting. Phosphate buffer saline (PBS), PBS+SB203580 (p38 MAPK inhibitor), PBS+MA, ISO, ISO+SB203580, ISO+MA were added to the primary cultured rat fibroblasts. Cells were collected after 48 h for subsequent detection. Results: In this study, the mouse model of myocardial fibrosis was successfully established. The left ventricular faction shortening (FS) and maximum rate of rise and maximum rate of fall of pressure in left ventricular chamber (±dp/dt) of the ISO+MA group were significantly higher than those of the ISO group ((35.1±1.8)% vs (28.5±2.6)%, (7 256±153) mmHg/s vs (6 402±240) mmHg/s, (7 156±163) mmHg/s vs (6 319±219) mmHg/s, all P<0.05). The levels of interstitial and perivascular collagen deposition in the ISO+MA group were higher than those in the ISO group (P<0.05), the relative mRNA levels of COL-1, COL-3 and TGF-β in the ISO+MA group were significantly lower than those in the ISO group, with the relative expression levels of 1.70±0.24 vs 3.69±0.34, 1.72±0.56 vs 4.84±0.82, 1.52±0.19 vs 2.64±0.29, respectively (all P<0.05). The phosphorylation levels of p38 MAPK, Smad3 and protein expression level of TGF-β1 in ISO+MA group were lower than those in ISO group (relative expression levels were 1.67±0.35 vs 2.61±0.58, 1.68±0.23 vs 2.52±0.19,1.56±0.15 vs 2.48±0.26, respectively, all P<0.05). The results of in vitro cell experiments showed that the mRNA levels of COL-1, COL-3 and TGF-β in the SB203580 and MA groups were significantly lower than those in the ISO group (relative expression levels were 2.25±0.51, 2.16±0.48 vs 5.29±1.21; 1.58±0.34, 1.69±0.29 vs 4.97±1.32; 1.41±0.31, 1.55±0.38 vs 3.53±0.56, respectively, all P<0.05). The phosphorylation levels of p38 MAPK and Smad3 in the SB203580 MA groups was significantly lower than those in the ISO group, and the protein expression level of TGF-β1 was lower than that in the ISO group (1.81±0.18, 1.77±0.16 vs 2.56±0.32; 1.85±0.21, 1.81±0.17 vs 2.48±0.37; 1.84±0.24, 1.72±0.17 vs 2.52±0.29, all P<0.05). Conclusion: Maslinic acid can inhibit the phosphorylation of p38 MAPK, thereby preventing the canonical TGF-β1/Smads fibrosis signaling pathway to achieve an anti-fibrosis role.

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.

The Therapeutic Effect and Mechanism of Static Progressive Stretching in Different Durations on Traumatic Knee Contracture in Rats

To investigate the effect and mechanism of static progressive stretching (SPS) in different durations on traumatic knee contracture in rats. Seventy male Wistar rats were randomly divided into three groups, including surgical modeling group ( n=50), control group (CON, no surgery, no treatment, n=10) and trauma without immobilization group (TRA, no treatment, n=10). The knee contracture model was established, and 50 surgical modeling rats were randomly divided into five groups including static progressive stretching treatment for 20 minutes group (S20 min, n=10), treatment for 30 minutes group (S30 min, n=10), treatment for 40 minutes group (S40 min, n=10), untreatment group (UNT, no SPS, n=10) and modeling group (MOD, n=10, euthanized after immobilization for histological staining and Western blot). Individuals in the S20 min, S30 min, and S40 min groups were anesthetized and submitted to SPS. One treatment session took place every other day. A total of 8 sessions were given till the final treatment session on the day 16. On the day 0, 8, and 16 of intervention, the range of joint motion (ROM) and gait analysis were measured and compared. After the ROM measurements and gait analysis, the rats were euthanized on the day 16 and the samples were stained with HE and Masson methods. The changes of pathological organization were observed. Western blot was used to detect the expressions of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6). ① ROM：the ROM of S30 min group recovered similar to that of the S20 min and S40 min groups after 8 days of treatment ( P>0.05), and was the best among all the surgical modeling groups after 16 d of treatment ( P<0.05). The ROM of S20 min, S30 min and S40 min groups significantly improved on the day 8 and day 16 comparing with that on day 0 ( P<0.01). ② Gait analysis: the stands in the S30min group improved best on the day 8 and day 16 ( P<0.05) , and better than that on day 0 ( P<0.05). The stride length of the S30 min group progressed similar to that of the S40 min group on the day 8 ( P>0.05), and there was no difference among three groups on the day 16 ( P>0.05). The stride length of the S30 min group appeared to recover more quickly on the day 8 ( P<0.05), and those of S20 min and UNT groups recovered significantly on the day 16 ( P<0.05). In addition, the swings in the S30 min group improved best ( P<0.05), and it appeared to recover better on the day 16 ( P<0.05). There was no statistical difference in terms of the swing speed among the four surgical modeling groups on the day 8 ( P>0.05). The swing speed of the S30min group increased most than those of the other three groups ( P<0.05), and it was much better on the day 8 and day 16 comparing with that on the day 0 ( P<0.05 ). ③ HE and Masson staining: the fibrosis and inflammation of the S30min group were significantly suppressed comparing to the other groups on the day 16. ④ Western blot: The protein expression levels of TGF-β1 and IL-6 were significantly lower than those in the other intervention groups including the S20 min, S40 min and UNT groups on the day 16 ( P<0.05). Static progressive stretching treatment for 30 min could significantly improve the traumatic knee contracture in rats. The mechanism may be that the SPS decreased the expressions of TGF-β1 and IL-6, reduced the adhesion and inflammation of joint capsule. Therefore it relieved the pain and increased the joint mobility by reconstructing the structure of the capsule and suppressing the fibrotic changes.

Role of miR-133a in regulating TGF-β1 signaling pathway in myocardial fibrosis after acute myocardial infarction in rats.

The aim of this research was to explore the effect of microRNA-133a (miR-133a) on myocardial fibrosis and cardiac function after myocardial infarction in rats, and to investigate the possible regulatory mechanism. Myocardial infarction model was successfully established in rats by ligation of the left anterior descending coronary artery. After miR-133a overexpression in rats myocardium, cardiac function was examined by echocardiography. Meanwhile, the degree of myocardial fibrosis was detected by Masson staining. In addition, the expression of α-smooth muscle actin (α-SMA) in cardiomyocytes was detected by immunohistochemistry. Quantitative Real-time polymerase chain reaction (qRT-PCR) was performed to analyze the expression level of miR-133a in the junction of myocardial infarction. The mRNA expressions of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), collagen type 1 (col 1), collagen type 3 (col 3) and α-SMA were measured by qRT-PCR as well. Furthermore, the protein levels of the above genes were detected by Western blotting. MiR-133a expression in the infarct border zone of myocardial tissue was significantly decreased on the 28th day after myocardial infarction surgery (p<0.05). In addition, up-regulation of miRNA-133a in myocardial tissue of rats with myocardial infarction could remarkably improve cardiac function and reduce collagen volume fraction. Furthermore, the mRNA and protein expression levels of TGF-β1, CTGF, col1, col3, α-SMA in myocardial tissue were obviously decreased after miRNA-133a up-regulation (p<0.001). Overexpression of miR-133a down-regulates the mRNA and protein levels of TGF-β1 and CTGF after myocardial infarction. Moreover, this may eventually reduce myocardial collagen deposition, inhibit myocardial fibrosis and improve cardiac function.

Effects Of Aerobic Exercise On Learning And Memory Ability And Hippocampal Tgf- 1 In Depressed Rats

OBJECTIVE: To investigate the effects of different periods of aerobic exercise intervention on hippocampal TGF-β1 expression and spatial learning and memory ability in depressive model rats. METHODS: 36 male SPF grade Sprague Dawley (SD) rats were randomly allocated into 4 groups (n=9) :Control group (C group), Pre-model exercise group (EC group), Model group (M group), Motion group(ce group). Except for group C, all other groups used CUMS stress stimulation to establish an animal model. Morris water maze test was used to detect spatial learning and memory ability of rats. Nissl staining was used to observe the morphology of rat hippocampal neurons. Real-time PCR, Western Blotting were used to detect the expression of TGF-β1. RESULTS: There was no significant difference in time-consumption of escaping the incubation period between the 1st and the 2nd day in each group (P > 0.05). On the fourth day, the escape latency of M group was significantly longer than that of rats in the C and ce groups (P < 0.05). On the 5th day, the escape latency in M group was significantly longer than that in C and ce groups (P < 0.05), and EC group was significantly longer than that in C group (P < 0.05). Results of the number of crossing platforms: The number of crossing in the M and EC groups were significantly lower than those in the C and ce groups (P < 0.01). Compared with EC and M group, the morphological structure of neurons in ce group was better.The expression of TGF-β1 mRNA in hippocampus of EC and ce group was significantly lower than that of C and M groups (P < 0.01). The expression levels of TGF-β1 protein in C and M groups were significantly higher than those in ce group (P < 0.01). The expression of TGF-β1 protein in M rats was significantly higher than that in EC and ce groups (P < 0.01). CONCLUSION: Aerobic exercise intervention can improve the morphological structure of hippocampal CA3 neurons, and decrease the expression of TGF-β1. It indicated that aerobic exercise can improve the depression.

Puerarin alleviates liver fibrosis via inhibition of the ERK1/2 signaling pathway in thioacetamide-induced hepatic fibrosis in rats.

Liver fibrosis is a complex pathological process and an early step in the progression of liver cirrhosis, which can eventually develop into hepatocellular carcinoma. Currently, there is no effective treatment for liver fibrosis. Puerarin is a traditional Chinese herb, which is commonly used in the treatment of various diseases. In addition, it is also believed to have a therapeutic effect in liver fibrosis. However, whether puerarin reduces liver fibrosis via the ERK1/2 signaling pathway to inhibit the activation of hepatic stellate cell (HSC) and excessive collagen deposition in liver fibrosis remains unknown. The aim of the current study was to establish a liver fibrosis in vivo model by intraperitoneal injection of thioacetamide (TAA) and investigate the effect of puerarin in the treatment of liver fibrosis. Hematoxylin and eosin and Van Gieson's staining were used to examine histopathological changes associated with liver fibrosis. Liver hydroxyproline content was examined to determine the total amount of collagen in the liver. The relative protein expression levels of transforming growth factor β1 (TGFβ1), α-smooth muscle actin (α-SMA), collagen type I, fibronectin, ERK1/2 and p-ERK1/2 were determined by western blot analysis. In the TAA group, histopathological changes and collagen fiber content in rat liver tissue samples were significantly increased compared with the control group (P<0.05). In addition, treatment with puerarin significantly decreased histopathological changes and collagen fiber content in rat liver tissue samples (P<0.05). The relative protein expression levels of TGFβ1, α-SMA, collagen type I, fibronectin and p-ERK1/2 were significantly upregulated in the TAA group compared with the control group (P<0.05), whereas puerarin treatment reversed these changes. These findings suggest that treatment with puerarin may reduce HSC activation and alleviate extracellular matrix protein expression levels by inhibiting the TGF-β/ERK1/2 pathway in liver fibrosis.

Long non-coding RNA snaR is involved in the metastasis of liver cancer possibly through TGF-β1.

It was previously demonstrated that the long non-coding RNA (lncRNA) small NF90-associated RNA (snaR) served an oncogenic role in human colon cancer, although its roles in other types of cancer remain unknown. To investigate the potential involvement of lncRNA snaR in hepatocellular carcinoma (HCC), expression of snaR in liver biopsies and plasma of patients with HCC and healthy controls was detected by reverse transcription-quantitative polymerase chain reaction. ELISA was used to determine the protein expression levels of transforming growth factor-β1 (TGF-β1). A snaR expression vector was transfected into HCC cells, and the effects on cell migration and invasion were analyzed by Transwell migration and Matrigel invasion assays, respectively. The protein expression levels of TGF-β1 in HCC cells were detected by western blotting. The expression of snaR and TGF-β1 was significantly increased in the patients with HCC compared with the healthy controls. The plasma expression levels of snaR and TGF-β1 were positively correlated in patients with HCC; however, not in healthy controls. snaR overexpression significantly promoted cancer cell migration and invasion, and additionally increased TGF-β1 expression. Treatment with TGF-β1 did not significantly affect snaR expression. A TGF-β1 inhibitor attenuated the effects of snaR overexpression in cancer cell migration and invasion. snaR may promote the metastasis of liver cancer through TGF-β1.

Effects of blueberry on hepatic fibrosis and expression of nuclear transcription factor-кB in rats

Objective: To observe the effects of blueberry and nuclear expression of transcription factor-кb (NF-кb) p65 in an experimental rat model of liver fibrosis. Methods: Forty-five Sprague-Dawley rats were randomly divided into isotonic saline control group (A); model group (B); blueberry juice prevention group (C, 15 g/kg); dan-shao-hua-xian capsule prevention group (D, 1 g/kg); and blueberry juice + dan-shao-hua-xian capsule prevention group (E). Rat liver fibrosis model was established by covalent compound carbon tetrachloride (CCl(4)). Each prevention group was given the corresponding dose of blueberry juice or (and) dan-shao-hua-xian capsule, and the rats were sacrificed after 8 weeks. The degree of liver fibrosis was evaluated by hematoxylin and eosin stain. A liver tissue of NF-κBp65 was detected by immunohistochemical method. The NF-κBp65 protein expression of liver tissue and transforming growth factor (TGF) β1 was detected by Western blot. Data of multiple groups were compared by one-way analysis of variance, and rank sum test. Results: Immunohistochemistry detected that TGFβ1 protein was mainly expressed in mesenchymal origin of hepatic stellate cells. The expression level of group A (3.75 ± 1.67) was low, while those of group B (9.00 ± 2.07), C (7.33 ± 1.00), D (6.00 ± 1.51), and E (3.5 ± 1.41) were high. However, the expression level of TGF-β1 protein in hepatic tissues of group B was significantly higher than that of group C, D and E [group E: 3.5 ± 1.41, F = 18.350, P < 0.05]. In addition, group D was higher than group E (P < 0.05). The expression of NF- kappa Bp65 protein was very complex, and the expression patterns in different groups were different (Statistical calculation of experimental data were based on expression of liver cells). Compared with group B (4.37 ± 2.13), the relative expression levels of NF-κBp65 protein in-group A (0.46 ± 0.25), group C (2.76 ± 1.01), group D (2.13 ± 1.51), group E (1.88 ± 0.99) were significantly decreased (F = 27.490, P < 0.05), and the expression trend was consistent with TGFβ1 protein. Western blot detected NF-κBp65 protein in liver tissues of rats. Compared with group A, levels in groups B, C, D and E were significantly increased (F = 96.983, P < 0.05), and groups C, D and E were significantly lower. The E group was significantly lower than the C group (F = 96.983, P < 0.05), and the degree of hepatic fibrosis was lower in each prevention group than in the B group (T = 24.1, P < 0.05). Conclusion: Blueberries have preventive effect on CCl4-induced hepatic fibrosis in rats, and its preventive mechanism may inhibit the expression and activation of NF-κBp65 in hepatocytes, thereby reducing TGFβ1- mediated production or activation.

Regulatory role of microRNA-185 in the recovery process after ankle fracture.

The present study aimed to investigate the expression of microRNA (miR)-185 in the bone and blood tissues following ankle fracture, and its regulatory mechanism in the ankle fracture recovery process. In total, 28 patients with ankle fractures were included, including 15 cases receiving surgical treatment within 1-7 days after fracture, and 13 cases receiving surgery within 8-14 days after fracture. Reverse transcription-quantitative polymerase chain reaction was performed to detect the mRNA expression levels. Western blot analysis and ELISA were used to determine the protein expression levels. Bioinformatics analysis and dual-luciferase reporter assay were applied to predict and confirm the upstream regulator of tumor growth factor (TGF)-β1. An MTT assay was performed to assess the cell proliferation. Compared with the 1-7-day surgery group, the mRNA and protein expression levels of TGF-β1 were significantly elevated, while the expression levels of miR-185 were significantly declined in the bone and blood tissues in the 8-14-day surgery group. Bioinformatics analysis and dual-luciferase reporter assay predicted and confirmed that TGF-β1 was the direct target gene of miR-185. Moreover, upregulated expression of miR-185 significantly decreased the protein expression levels of TGF-β1 and reduced the proliferating activity of hFOB1.19 cells. Within two weeks after ankle fracture, the expression levels of TGF-β1 are significantly upregulated in the bone and blood tissues, which may have been associated with the downregulated expression of miR-185. miR-185 may modulate TGF-β1 to regulate the recovery of ankle fracture. These findings may contribute to the understanding of the biological functions and effects of miRNA-185 and TGF-β1 in ankle fractures.