TGF-β1 Protein Expression Research Articles

Mesenchymal Stem Cell-conditioned Medium Attenuated CoCl2-induced Injury of Renal Tubular Epithelial Cells by Inhibiting NCOA1, HIF-1α, and Sox9.

Renal interstitial fibrosis (RIF) constitutes the ultimate pathological alteration in nearly all chronic kidney diseases (CKD). Mesenchymal stem cell conditioned medium (MSC-CM) exhibits an alleviating impact on renal fibrosis; however, the underlying mechanism remains unclear. The objective of this study was to explore whether MSC-CM regulates the expression of α-smooth muscle actin (α-SMA), Transforming growth factor-β1 (TGF-β1), Hypoxia-inducible factor-1α (HIF-1α), Nuclear receptor coactivators (NCOA1), and SRY-related high mobility (Sox9). Rat renal tubular epithelial cells (RTECs), NRK-52E, were treated with diverse concentrations of Cobalt chloride (CoCl2) for 24 hours. The survival rate and protein expression of NRK-52E cells exposed to different concentrations of CoCl2 were determined to identify the final concentration. Three groups of NRK-52E cells were employed in the experiment: the normal control group, the 400 μM CoCl2 group, and the MSC-CM + 400 μM CoCl2 group. The cell morphology was observed by an inverted phase contrast microscope and scanning electron microscope, and the protein expressions of α-SMA, TGF-β1, HIF-1α, NCOA1, and Sox9 were detected. The microscopic findings demonstrated that MSC-CM was able to decrease the degree of cytochemical hypoxia damage in NRK-52E cells induced by CoCl2. Immunofluorescence and Western blot analyses also affirmed a similar tendency. The upregulation of α-SMA, TGF-β1, HIF-1α, NCOA1, and Sox9 triggered by CoCl2 could be inhibited following MSC-CM intervention. Our findings indicate that MSC-CM exerts a protective effect on RTECs by down-regulating α-SMA, TGF-β1, HIF-1α, NCOA1, and Sox9.

Unraveling TGF-β1's Role in Mediating Fibrosis and Cell Death in Feline Kidney Cells.

Chronic kidney disease (CKD) is prevalent among older cats. The transforming growth factor beta 1 (TGF-β1) pathway is associated with renal fibrosis. TGF-β1 signaling through the non-canonical/smad-independent pathway activates mitogen-activated protein kinase (MAPK) signaling, which is linked to fibrosis and apoptosis. The MAPK pathway regulates the Bcl-2 protein family, which is known for its anti-apoptosis properties. This study aimed to quantify the mRNA expression of the TGFβ, MAPK, and Bcl2 genes and the protein expression of TGF-β1 and MAPK in feline kidney cells and tissue. A gene expression analysis was conducted using qPCR to calculate the relative gene expression, while the protein expression was assessed through Western blot analysis. Immunohistochemistry staining of TGF-β1 and MAPK was performed on feline kidney tissue. The results revealed the significant upregulation of TGFβ (p = 0.001) and considerable downregulation of Bcl2 (p = 0.010) in doxorubicin-treated feline kidney cells. The immunostaining levels of TGF-β1 and MAPK were higher in the kidney tissue of cats with CKD than in non-CKD cats. However, there was no difference in TGFβ, MAPK, or Bcl2 gene expression in CKD vs. non-CKD cats. The findings suggest that TGF-β1 and Bcl-2 are associated with renal fibrosis and apoptosis in feline kidney cells. A deeper understanding of the TGF-β1 pathway could enable veterinarians to monitor disease progression and mitigate complications in feline CKD.

The effect of tocilizumab treatment for skin fibrosis by inhibiting CD38+ macrophages in systemic sclerosis.

Dermal and pulmonary fibrosis are the main clinical symptoms of systemic scleroderma (SSc), for which there are no effective therapeutic agents. Tocilizumab is thought to improve the symptoms of fibrosis, but the effect of tocilizumab on dermal fibrosis has not been explored. This study aims to investigate the therapeutic effect of tocilizumab on skin fibrosis by inhibiting CD38+ macrophages in the bleomycin-induced SSc mice model. The 8-week-old BALB/c mice were randomly divided into three groups: control group (PBS group), model group (BLM group), and tocilizumab group (TCZ group). The mRNA expression of VIMENTIN, TIMP1, and COL1A1 was measured by qPCR. Western blot was used to detect the protein expression of α-SMA, TGF-β, and COL1A1 in skin tissues. The expression of CD38+ macrophages in the BLM-induced fibrosis mouse model was verified by flow cytometry and immunofluorescence. In comparison to the PBS control group, mice in the BLM group showed skin fibrosis, edema, thickness, and collagen deposition. The percentage of macrophages in the skin, peripheral blood, and spleen was significantly increased in the BLM group, and the percentage of CD38+ macrophages increased in the skin and peripheral blood but decreased in the spleen. After co-cultured with macrophages, L929 fibroblasts differentiated into myofibroblasts, with increased mRNA expression of COL1A1, COL3A, TGF-β, and Fibronectin. Furthermore, after being stimulated by LPS, RAW264.7 cells showed increased expression of IL-6 and CD38. The mRNA levels of COL1A1, COL1A2, COL3A, TGF-β, and Fibronectin in L929 fibroblasts were markedly increased when co-cultured with LPS-stimulated RAW264.7 cells. Tocilizumab treatment reduced dermal thickness and collagen deposition induced by BLM. Furthermore, the percentage of total macrophages and CD38+ macrophages in the skin and peripheral blood significantly decreased after tocilizumab treatment. This study revealed that tocilizumab improved skin fibrosis in the SSc mice model, which was mediated by inhibiting skin and peripheral CD38+ macrophages.

Duhuo Jisheng Decoction in reduction of inflammatory response via Transforming growth factor-β/Smad signaling pathway for repairing rabbit articular cartilage Injury: A Randomized Controlled Trial

ObjectiveThis study aims to investigate the mechanism underlying the effect of Duhuo Jisheng Decoction on the repair of rabbit articular cartilage injury through a reduction in the inflammatory response mediated by the Transforming growth factor (TGF)-β/Smad signaling pathway. MethodsA rabbit articular cartilage injury model was constructed using a ring bone extraction drill. Twenty-four Japanese white rabbits were randomly divided into six groups, namely Sham operation, model, low-dose Duhuo Jisheng Decoction, medium-dose Duhuo Jisheng Decoction, high-dose Duhuo Jisheng Decoction, and positive control groups. The treatment lasted 12 weeks. Gross observation, International Cartilage Repair Society score, Wakitani score, and Micro-computed tomography analysis were used to evaluate the structural repair of cartilage injury. Histology and immunohistochemistry were used to observe the proteoglycan, P-TβRII, P-Smad2, and type II collagen expression levels. Enzyme-linked immunosorbent assay was used to analyze the concentrations of Matrix Metalloproteinase-13 and Syndecan-4 in the joint fluid; and RT-PCR and Western Blot were used to observe the mRNA and protein expressions of ALK5, Sox-9, P-Smad3, and TGF-β1 at the injury repair site. ResultsThe repair effect of cartilage injury, as seen through gross observation and quantitative scoring, was better in all the Duhuo Jisheng Decoction treatment groups than in the model group. The medium dose group of Duhuo Jisheng Decoction had the best repair effect. We observed remarkable structural restoration of cartilage injury in the medium-dose Duhuo Jisheng Decoction group, with the subchondral bone presenting a distinct hierarchy, and parameters such as bone volume fraction and trabecular separation/spacing being significantly augmented. We found high expression levels of proteoglycans, P-TβRII, P-Smad2, and type II collagen. The concentrations of Matrix Metalloproteinase-13 and Syndecan-4 in the joint fluid were significantly lower following treatment. The low gene expression levels of ALK5, Sox-9, P-Smad3, and TGF-β1 in the injury site of the model group could be reversed in the medium-dose Duhuo Jisheng Decoction group. ConclusionDuhuo Jisheng Decoction can repair rabbit cartilage injury and reverse the levels of inflammatory factors in the joint fluid. The mechanism underlying its therapeutic effect is related to the activation of the TGF-β/Smad signaling pathway. This study provides a reliable basis for using Duhuo Jisheng Decoction to treat cartilage injury following knee osteoarthritis.

Impacts of mesenchymal stem cells and hyaluronic acid on inflammatory indicators and antioxidant defense in experimental ankle osteoarthritis.

No effective treatment guarantees full recovery from osteoarthritis (OA), and few therapies have disadvantages. To determine if bone marrow mesenchymal stem cells (BMMSCs) and hyaluronic acid (HA) treat ankle OA in Wistar rats. BMMSCs were characterized using flow cytometry with detection of surface markers [cluster of differentiation 90 (CD90), CD105, CD34, and CD45]. Fifty male Wistar rats were divided into five groups of 10 rats each: Group I, saline into the right tibiotarsal joint for 2 days; Group II, monosodium iodate (MIA) into the same joint; Groups III, MIA + BMMSCs; Group IV, MIA + HA; and Group V, MIA + BMMSCs + HA. BMMSCs (1 × 106 cells/rat), HA (75 µg/rat), and BMMSCs (1 × 106 cells/rat) alongside HA (75 µg/rat) were injected intra-articularly into the tibiotarsal joint of the right hind leg at the end of weeks 2, 3, and 4 after the MIA injection. The elevated right hind leg circumference values in the paw and arthritis clinical score of osteoarthritic rats were significantly ameliorated at weeks 4, 5, and 6. Lipid peroxide significantly increased in the serum of osteoarthritic rats, whereas reduced serum glutathione and glutathione transferase levels were decreased. BMMSCs and HA significantly improved OA. The significantly elevated ankle matrix metalloproteinase 13 (MMP-13) mRNA and transforming growth factor beta 1 (TGF-β1) protein expression, and tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) serum levels in osteoarthritic rats were significantly downregulated by BMMSCs and HA. The effects of BMMSCs and HA on serum TNF-α and IL-17 were more potent than their combination. The lowered serum IL-4 levels in osteoarthritic rats were significantly upregulated by BMMSCs and HA. Additionally, BMMSCs and HA caused a steady decrease in joint injury and cartilage degradation. BMMSCs and/or HA have anti-arthritic effects mediated by antioxidant and anti-inflammatory effects on MIA-induced OA. MMP-13 and TGF-β1 expression improves BMMSCs and/or HA effects on OA in Wistar rats.

Phyllanthi Fructus ameliorates hyperuricemia and kidney injure via inhibiting uric acid synthesis, modulating urate transporters, and alleviating inflammation

Phyllanthi Fructus, known as Yuganzi (YGZ), is a unique medicine and food homologous fruit with both medicinal and nutritional properties. Its historical use in treating hyperuricemia (HUA) and gout is well-documented. However, the precise therapeutic effects and potential molecular mechanisms remain unclear. In this study, an experimental rat modelling by a high-fat/high-sugar diet and potassium oxonate/adenine oral administration was used to evaluate the pharmacodynamic effects of YGZ. Network pharmacology, molecular docking and molecular dynamics simulation were utilized to elucidate the potential mechanisms. Supplementation with YGZ effectively ameliorated HUA by inhibiting xanthine oxidase activity, and enhancing uric acid excretion through up-regulating of OAT1 and ABCG2, while down-regulating of URAT1. Furthermore, YGZ supplementation enhanced superoxide dismutase activity, reduced malondialdehyde content, and inhibited the secretion of IL1B, IL6, TNFα, ICAM1, VCAM1, TGFβ1, and NF-κB protein expression. Network pharmacology analysis indicated that YGZ influences 138 targets, modulating the disease network via lipid and atherosclerosis, insulin resistance, HIF-1, TNF, IL-17, TLRS, and NF-κB signaling pathways. Molecular docking analysis suggested that organic acids (e.g. ellagic acid, gallic acid) and flavonoids (e.g. quercetin, delphinidin, luteolin, epigallocatechin gallate) exhibited superior binding abilities to key targets (e.g. XDH, ABCG2, URAT1, OAT1, IRS1, PTGS2, TLR4). Noteworthy, molecular dynamics simulations confirmed that epigallocatechin gallate binds to URAT1 with the greatest stability. These results provide substantial evidence for the therapeutic efficacy of YGZ and establish a theoretical foundation for the development of natural products in treating hyperuricemia.

Effect of mesenchymal stem cell derived from umbilical cord blood on rabbit intrauterine adhesion model

Objective: To investigate the repair effect of human umbilical cord blood mesenchymal stem cells (hUCB-MSC) on endometrium of intrauterine adhesion (IUA) by establishing animal model. Methods: Eighteen healthy female New Zealand white rabbits were divided into a control group, an IUA group and an hUCB-MSC transplantation group according to random number table method. The control group underwent laparotomy only. The IUA group underwent IUA modeling surgery using curettage and lipopolysaccharide (LPS) cotton placed in uterine cavity for double injury. The hUCB-MSC transplantation group received the same IUA modeling surgery followed by multipoint injection of hUCB-MSC suspension (2×106 cells/ml, 500 μl) into the bilateral uterine myometrium one week after surgery. Four weeks after the IUA modeling surgery, the rabbits were euthanized and samples were collected. Hematoxylin and eosin (HE) staining and Masson staining were used to assess the number of endometrial glands and the fibrosis rate. RNA sequencing was performed on the endometrium of the IUA group and hUCB-MSC transplantation group.The mRNA and protein expression of the fibrosis markers [transforming growth factor β1 (TGF-β1) and tissue inhibitors of metalloproteinase 1 (TIMP1)] and RNA sequencing-related parameters were detected by quantitative real-time PCR (qRT-PCR) and Western blot. Results: Compared with the control group, the number of glands in IUA group decreased (26.33±1.53 vs 4.33±1.53, P<0.001), and the fibrosis rate increased (18.01%±2.21% vs 69.55%±2.42%, P<0.001), indicating successful modeling. HE and Masson staining revealed that the number of glands in the hUCB-MSC transplantation group was increased compared with that in IUA group (17.33±2.52 vs 4.33±1.53, P<0.001), and the fibrosis rate decreased (69.55%±2.42% vs 41.55%±1.99%,P=0.001). Western blot analyses of fibrosis-related proteins (TGF-β1 and TIMP1) showed elevated levels of TGF-β1 (0.91±0.05) and TIMP1 (0.99±0.01) proteins in the IUA group compared to control group (0.61±0.04, 0.68±0.07) (P=0.001, 0.015). The hUCB-MSC transplantation group exhibited reduced expression of TGF-β1 (0.69±0.04) and TIMP1 (0.62±0.08) proteins compared to the IUA group (P=0.005, 0.014). Western Blot results related to the PI3K/AKT signaling pathway [phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT)] showed that the expression of p-PI3K(1.05±0.05) and p-AKT(1.17±0.06) in the IUA group increased compared to the control group (0.78±0.03, 0.85±0.05) (P=0.002, 0.002). The expression of p-PI3K (0.74±0.02) and p-AKT (0.93±0.04) proteins in the hUCB-MSC transplantation group decreased compared to the IUA group (P=0.003, 0.005). Conclusion: hUCB-MSC can repair the damaged endometrium in rabbit intrauterine adhesion model by increasing the number of endometrial glands and improving its level of fibrosis.

Preclinical Evidence of Mulberry Leaf Polysaccharides on Diabetic Kidney Disease: a Systematic Review and Meta-Analysis.

Mulberry leaf polysaccharides (MLPs) have a variety of biological activities. Preliminary scattered evidence of preclinical studies have reported their potenzial effects on diabetic kidney disease (DKD). Here, we intended to assess the preclinical evidence of MLPs and explore their potenzial mechanisms on DKD, offering a scientific reference for the therapeutic use of MLPs. The study has been registered under the CRD42022309117 registration number at PROSPERO. Comprehensive search was conducted across eight databases from their establishment till January 2024, and eight studies with 270 animals were included in the meta-analysis. The primary outcome measurements in the MLP group, including serum creatinine (Scr) (P = 0.0005), blood urea nitrogen (BUN) (P = 0.02), 24-hour urinary protein (UP) (P = 0.001), and urinary microalbumin (UAlb) (P < 0.0001), were significantly reduced compared to the control group. Additionally, MLP treatment was significantly correlated with fasting blood glucose (FBG), total cholesterol (TC), protein expression of TGF-β1, CTGF mRNA, and the kidney index (all P values < 0.05) and delayed the progression of local pathological changes in the kidney. Subgroup analysis revealed significant species differences in the efficacy of MLPs. Also, it showed that the dosage of streptozotocin potenzially affected the Scr and UAlb results, while the duration of MLP treatment influenced UAlb results. MLPs may exert potenzial renal protection by delaying renal fibrosis, inhibiting inflammatory reactions, suppressing the growth hormone-insulin-like growth factor-insulin-like growth factor binding protein axis, and regulating the insulin receptor pathway. In summary, MLPs have multifaceted renal protective effects, suggesting their potenzial for treating DKD.

Mitochondrial Oxidative Stress and Cardiac Dysfunction in TERT Knockout Mice

Abstract Background Heart failure (HF) is a major cause of hospitalization in the elderly, with its incidence increasing due to aging and associated risk factors like hypertension, diabetes, and myocardial fibrosis. Aging-related myocardial fibrosis, characterized by alterations in the extracellular matrix and myocardial structure, impairs cardiac function. Mitochondrial oxidative stress plays a pivotal role in cardiac electrical and structural remodeling, necessitating a deeper understanding of its involvement in aging-related heart failure. Purpose The purpose of this study is to investigate the mechanisms of aging-related ventricular electrical and structural remodeling by constructing an aging mouse model. This research aims to provide potential therapeutic targets for heart failure associated with aging. Methods We developed a third-generation homozygous telomerase reverse transcriptase (TERT) knockout mouse model. Comparing third-generation homozygous TERT knockout mice (F3 group) with age-matched wild-type males (WT group). The study encompassed blood pressure measurement, electrocardiographic analysis, echocardiography, ELISA for cardiac biomarkers, ventricular tissue electrophysiology, fibrosis assessment through staining, transcriptomic profiling via RNA-seq, and electron microscopy of ventricular mitochondria. Results Compared to the WT group, the F3 group demonstrated increased P53 and P16 protein expression. Decreased LVEF and FS, along with increased interventricular septum thickness, left ventricular diameter, and left ventricular volume. Elevated serum NT-pro-BNP and troponin I (cTnI) levels, with prolonged QRS duration. Decreased left and right ventricular conduction velocity, accompanied by increased conduction dispersion. Notable elevation in COLI, TGFβ, and α-SMA gene transcription and protein expression in ventricular tissue. Significant differences in mitochondrial oxidative stress signal pathways via RNA-seq analysis. Elevated serum MDA levels and decreased SOD levels, with up-regulated Mn-SOD gene transcription and protein expression, and down-regulated MFN2 and Drp1 gene transcription and protein expression in ventricular tissue. Electron microscopy revealed mitochondrial abnormalities such as loose arrangement, decreased number, swelling, vacuolation, and myofilament disintegration or breakage in the F3 group. Conclusion Third-generation TERT-/- senescent mice exhibit notable cardiac impairments, including electrical remodeling, structural changes, and mitochondrial dysfunction. These findings suggest a potential link between mitochondrial oxidative stress and aging-related heart failure, indicating novel therapeutic opportunities targeting mitochondrial dysfunction for managing such conditions.

Transforming growth factor-beta and Epithelial-mesenchymal transition is considered an attractive pivotal Genetic-Biomarker for Hepatocellular carcinoma with hepatitis C virus-infection patients

Abstract Introduction/Objective Hepatocellular carcinoma (HCC) is the most common cause of cancer worldwide. HCC is induced by hepatitis C-virus (HCV) which represents a major health problem in Egypt. The transforming growth factor- beta (TGF-β) is a potent cytokine that has a multifunctional pro-fibrotic activity, for enhancing liver-inflammation, fibrosis, tumor-progression, and metastasis. TGF-β has a vital role in the regulation of tumorigenesis, by controlling epithelial-mesenchymal-transition (EMT), apoptosis, proliferation, and differentiation process. The dysregulation of TGF-β, Wnt/β-catenin signaling pathways, by HCV-core protein is implicated in the development of HCC. Methods/Case Report The study aims to determine the effect of the HCV-infection in HCC patients with a set of growth-factors, EMT, and anti-apoptotic protein expression levels by using western blotting and immunohistochemistry staining analysis. Results (if a Case Study enter NA) These results observed an insignificant increase in the expression of TGF-β protein for HCC/HCV-infected cells compared to the HCV-uninfected cells, and the expression of the β-catenin protein level is elevated in the nucleus. Also, it showed that Bcl-2 protein is a significant increase in HCV-infected when compared to HCV-free groups. Positive staining for p53 protein was detected in the HCC/HCV-infected, more than in HCC/HCV-uninfected cells. This data indicated that the core-protein of HCV can switch TGF-β from a tumor suppressor to a tumor promoter by suppressing apoptosis of hepatocytes and increasing EMT expression by activation of Wnt/β-catenin signaling pathway. Therefore, HCV-core protein enhances the nuclear accumulation of β-catenin expression protein to activate downstream target genes, which regulate cell-growth and cell-cycle progression. Conclusion It is speculated that HCV may exert an anti-apoptotic effect, and thus enhance tumorigenesis. Also, the accumulation of nuclear Wnt/β-catenin and p53 correlates with TGF-β expression, typically a late event in hepato- carcinogenesis. Taken together, the expression level of TGF-β, Wnt/β-catenin, Bcl-2, and p53 genotype in HCV is considered an indicator of shortened survival in patients, and may be an attractive clinically pivotal genetic biomarker to mediate liver pathogenesis.

Preventive Effect of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole on Pulmonary Fibrosis through Inhibition of Inflammation and Down-regulation of TGF-b Signaling Pathway.

In the present study effect of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on plmonary fibrosis development was investigated in vitro in primary lung fibroblasts as well as in vivo in mice model. The results demonstrated that STT treatment effectively inhibited the TGF-β1 induced increase in expression of α-SMA and collagen I proteins in PLFs. STT treatment effectively reversed the TGF-β1 induced increase in expression of LOXL2 protein and phosphorylation of Smad2/3 proteins. Treatment of PLFs with STT reversed the TGF-β1-induced increase in expression of NOX4 and suppression of p-AMPK protein. In mice model of pulmonary fibrosis STT treatment significantly inhibited the BLM-mediated decrease in body weight and survival rate. The BLM induced increase in pulmonary index in mice was also effectively inhibited on treatment with STT. Treatment of the mice with STT inhibited the BLM-induced increase in α-SMA and Col I protein expression in pulmonary tissues. The BLM-induced increase in TGF-β1 protein expression in pulmonary tissues of the mice was inhibited on treatment with STT. Treatment with STT effectively promoted the AMPK activation in lung tissues of the BLM administered mice. In summary, the present study demonstrates that STT treatment prevents TGF-β1 induced up-regulation of α-SMA, collagen I, LOXL2 protein expression and targets phosphorylation of Smad2/3 proteins in PLFs. Moreover, it inhibits TGF-β1-induced increase in expression of NOX4 and reverses TGF-β1-mediated suppression in expression of p-AMPK protein. Therefore, STT inhibits fibrosis development in vitro as well as in vivo and therefore can be investigated further as a therapeutic agent for the treatment of lung fibrosis.

Effect of Buzhong Yiqi Decoction on Treg cells in rats with autoimmune thyroiditis through TGF-β/Smad signaling pathway

To investigate the effect of Buzhong Yiqi Decoction on regulatory T cells(Treg) in experimental rats with autoimmune thyroiditis(EAT) through the transforming growth factor-β(TGF-β)/Smad signaling pathway. Female SD rats were immunized with iodine-rich drinking water combined with Freund's adjuvant and porcine thyroglobulin(pTG) to establish the EAT model of rats, and the levels of serum thyroperoxidase antibody(TPOAb) and thyroglobulin antibody(TGAb) were detected. Pathological sections by hematoxylin-eosin(HE) staining were observed. Treg in the rats' spleen were extracted by immunomagnetic beads after the successful modeling and identified by flow cytometry. The extracted Treg were divided into blank group, Buzhong Yiqi Decoction group, TGF-β group, antagonist(LY3200882), and antagonist(LY3200882)+Buzhong Yiqi Decoction group. After the intervention, the cell counting kit-8(CCK-8) experiment was conducted to detect cell viability. Western blot and quantitative real-time PCR(RT-qPCR) were used to detect the expression of TGF-β/Smad signaling pathway-related proteins and genes. The results showed that the levels of TPOAb and TGAb increased in the rats in the model group compared to the rats in the blank group. HE staining showed that part of the follicles in the thyroid tissue of the rats in the model group were destroyed, and a large number of lymphocytes were infiltrated, indicating that the modeling was successful. After Treg were administered in vitro, CCK-8 results showed that the serum concentration of Buzhong Yiqi Decoction was below 40% to promote cell proliferation. The Buzhong Yiqi Decoction-containing serum group could increase the protein expression of TGF-β1, FoxP3, Smad2, and Smad4 compared with the blank serum group, while the expression of p-Smad2, p-Smad3, and Smad3 increased compared with the blank serum group, but the difference was not statistically significant. Compared with the antagonist group, the protein expressions of p-Smad2, Smad2, p-Smad3, Smad3, Smad4, and Smad7 did not significantly increase or decrease in the antagonist group after adding Buzhong Yiqi Decoction-containing serum. RT-qPCR showed that compared with the blank serum group, the mRNA expression of TGF-β1, FoxP3, Smad2, Smad3, Smad4, and Smad7 in the Buzhong Yiqi Decoction group increased or decreased in the same trend as that in the TGF-β group, but there was no statistical significance. After Buzhong Yiqi Decoction-containing serum was added to the antagonist group, the mRNA levels of TGF-β1, FoxP3, Smad2, Smad3, Smad4, and Smad7 were not statistically significant. In conclusion, Buzhong Yiqi Decoction could promote the stability and activity of Treg cells by promoting the secretion of TGF-β1 and regulating the expression of key signaling molecules TGF-β1, Smad2, and Smad4 in the TGF-β/Smad signaling pathway, thus affecting the immune balance of Th17/Treg and inhibiting the inflammatory response of rats with EAT.

The effects of extracorporeal shock wave therapy on cutaneous radiation injury in a mouse model.

Although radiation-induced skin injuries are a concern in patients receiving radiation therapy, there are few effective treatments. The aim of this study was to evaluate the protective effects of extracorporeal shock wave therapy (ESWT) on irradiated fibroblasts and mouse skin. In the in vitro study of human dermal fibroblasts, the experimental group was subjected to ESWT following irradiation (20 Gy). The control groups were only irradiated or only subjected to ESWT. At 24 or 48 h post-ESWT, cell viability, cell migration, and mRNA and protein expression were measured. In the in vivo study, the experimental group (7 mice) was treated with ESWT following irradiation (45 Gy). The control group (7 mice) was only irradiated. At 8 weeks post-irradiation, dorsal skin was harvested for histopathologic examination and protein isolation. In dermal fibroblasts, treatment with ESWT increased viability of irradiated cells compared with irradiated-only and untreated cells (p = 0.005). ESWT increased cell migration 24 h post-irradiation (p = 0.002), and decreased TGF-β1 protein expression 48 h post-irradiation (p = 0.024). In mice, ESWT decreased the level of radiation-related skin injury (p = 0.006). Treatment of irradiated skin with ESWT decreased TGF-β1 (p = 0.009) and phospho-Smad3 (p = 0.009) protein expression, as well as decreasing myofibroblasts (p = 0.047) and increasing vessel density (p < 0.001). Our study demonstrated that ESWT alleviated radiation-induced fibrosis by downregulating TGF-β1 expression. It suggests the potential of ESWT for the treatment of radiation-induced fibrosis.

CGS-21680 defers cisplatin-induced AKI-CKD transition in C57/BL6 mice

Acute kidney injury (AKI), with a high mortality and morbidity, is known as a risk factor for developing progressive chronic kidney disease (CKD). Targeting transition of AKI to CKD displays an excellent therapeutic potential. This study aims at investigating the role of CGS-21680, selective A2AR agonist, in deferring Cis-induced AKI-CKD transition. The AKI-CKD transition model was induced in C57/BL6 mice by repeated low doses of Cis (2.5 mg/kg i.p for 5 consecutive days in two cycles with a recovery phase of 16 days between two cycles). CGS-21680 was administered daily for 6 weeks (0.1 mg/kg, i.p). Urine, blood, and kidney were collected at three different time points to track the disease progression. CGS-21680 administration preserved kidney function and attenuated tubular damage as evidenced by hematoxylin-eosin (H&E) histopathology. CGS-21680 significantly restored oxidative status as reflected by reduced malondialdehyde (MDA) content and increased total antioxidant capacity (TAC). CGS-21680 showed anti-inflammatory effect as indicated by decreased TNF-α and iNOS. Additionally, CGS-21680 ameliorated endothelial dysfunction and enhanced renal vasodilation as evidenced by upregulation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) expression and down regulation of endothelin-1 (ET-1) and its receptor endothelin-A (ET-A) receptor expression. CGS-21680 also attenuated renal fibrosis as reflected by the reduction of percentage of fibrosis in Masson's trichome-stained renal sections and down regulation of transforming growth factor beta1 (TGF-β1) protein expression in IHC-stained renal sections. In conclusion, CGS-21680 could defer Cis-induced AKI-CKD transition via its vasodilatory, antioxidant, anti-inflammatory, and anti-fibrotic effects.

Hepatoprotective action mechanism and quantification of soyasaponin Bb in Abri Herba by HPLC and network pharmacology

Ethnopharmacological relevanceThe herb of Abrus cantoniensis Hance (AC) is an important Traditional Chinese Medicine (TCM) and is also used as an herbal tea with hepatoprotective action. Soyasaponin Bb is one of the pharmacodynamic substances of AC for the herb's effective pharmacological activity. This study aims to investigate the anti-fibrotic and hepatoprotective activities of soyasaponin Bb in vivo and in vitro experiments, mechanism by network pharmacology and quantification by HPLC. Materials and methodsHigh-performance liquid chromatography (HPLC) was applied to evaluate the quality of the herb and determine the contents of soyasaponin Bb from different sources and parts of the AC. In vivo experiments were conducted to induce an acute liver injury model by injecting CCl4 into mice, and an in vitro hepatic fibrosis model was established by cultivating LX-2 cells with TGF-β1. These models were used to explore the anti-fibrotic and hepatoprotective effects of soyasaponin Bb and its underlying mechanisms. In addition, the potential target genes corresponding to soyasaponin Bb were identified using the Swiss Target Prediction database through network pharmacology methods. Meanwhile, hepatic fibrosis targets were screened using the GeneCards, TTD, and OMIM disease databases. The STING database was used to construct the protein-protein interaction (PPI) network of soyasaponin Bb-hepatic fibrosis. The soyasaponin Bb-hepatic fibrosis disease target-pathway network was constructed using Cytoscape 3.9.1 software. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to enrich and analyze the common targets of the drug and the disease, aiming to identify the potential targets and pathways involved in the anti-fibrotic and hepatoprotective effects of soyasaponin Bb. ResultsThe content of soyasaponin Bb varied across different sources, with the roots containing the highest concentration, up to 0.2480%. In vivo experiments showed that soyasaponin Bb had a protective effect against CCl4-induced acute liver injury, effectively inhibiting the increase in ALT and AST levels and slowing down the hepatocyte inflammatory damage caused by CCl4. Soyasaponin Bb also down-regulated MDA levels and up-regulated SOD levels, indicating a certain antioxidant capacity. In vitro cell experiments showed that soyasaponin Bb could effectively inhibit the proliferation of HSC-LX2 cells induced by TGF-β1 by regulating the TGF-β1/α-SMA pathway, significantly down-regulate the protein expression of TGF-β1 and α-SMA, while also reducing the levels of inflammatory cytokines IL-6 and IL-1β. Further network pharmacology analysis suggested that soyasaponin Bb can exert anti-fibrosis activity by regulating the IBD signaling pathway, Th17 signaling pathway, Hepatitis B signaling pathway, and JAK-STAT signaling pathway. ConclusionSoyasaponin Bb is primarily distributed in the root of AC, and it has a strong protective effect against CCl4-induced acute liver injury. It can reduce the level of inflammatory factors, relieve inflammation, and exert anti-fibrotic activity by regulating the TGF-β1/α-SMA pathway. Its potential anti-hepatic fibrosis mechanism has been investigated through network pharmacology.

The mechanism of TGF-β mediating BRD4/STAT3 signaling pathway to promote fibroblast proliferation and thus promote keloid progression

ObjectiveThe purpose of this study was to investigate the effect of TGF-β on keloid and its molecular mechanism in fibroblasts. Methods: The difference between normal tissue and keloid tissue can be detected using HE staining. Fibroblasts were treated with TGF-β, and then treated with the BRD4 inhibitor JQ1 and the STAT3 activator Colivelin TFA. Western blot was used to measure the relative protein expression of TGF-β, BRD4, p-STAT3, p-EZH2, C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Immunofluorescence staining was used to measure the relative fluorescence intensity of BRD4, p-STAT3, α-SMA, and Collagen-I. Cell proliferation ability was evaluated by CCK-8 assay and colony formation assay. Results:The expression of TGF-β and BRD4 was significantly higher in keloid tissue compared to normal tissue. TGF-β mediated the BRD4/STAT3 signaling pathway to inhibit p-EZH2 and promote the expression of C-myc, KLF2, KLF4, α-SMA, and Collagen-I. Additionally, TGF-β mediated the BRD4/STAT3 signaling pathway to enhance fibroblast proliferation. Conclusion:TGF-β mediates the BRD4/STAT3 signaling pathway to promote fibroblast proliferation and contribute to the progression of keloid.

Extracorporeal Shock Wave and Melatonin Alleviate Joint Capsule Fibrosis after Knee Trauma in Rats by Regulating Autophagy.

Joint contracture is a common clinical problem affecting joint function. Capsule fibrosis plays a pivotal role in the progression of Joint contracture. Previous studies have reported that autophagy plays a regulatory role in visceral fibrosis. This study aimed to investigate whether extracorporeal shock wave therapy (ESWT) and melatonin alleviate joint capsule fibrosis in rats with extended knee joint contracture by regulating autophagy. A rat knee joint extension contracture model was made. Then, the rats were treated with ESWT, melatonin, ESWT + melatonin, or ESWT + melatonin + mTOR agonist for 4 weeks. The range of motion (ROM) of the knee joints was measured. Joint capsules were collected and observed for pathological changes by H&E and Masson staining. LC3B protein expression was evaluated by immunofluorescence staining. TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ, LC3, ATG7, Beclin1, p-AMPK, p-mTOR and p-ULK1 protein expressions were measured by Western blot assay. The intervention groups had significantly improved ROM of knee joint (P < 0.05), significantly improved pathological changes on HE and Masson staining, significantly decreased protein expressions of TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ and pmTOR (P < 0.05), and significantly increased protein expressions of LC3B, LC3II/LC3I ratio, ATG7, Beclin1, p-AMPK, and p-ULK1 (P < 0.05). Among these groups, the effects demonstrated by the ESWT + melatonin group were the best. With the mTOR agonist supplement, the therapeutic effects of extracorporeal shock waves and melatonin were significantly reduced. ESWT plus melatonin alleviated knee joint capsule fibrosis in rats by regulating autophagy.

Diterpene glycosides from Fructus Rubi ameliorates benign prostatic hyperplasia in rats through the androgen and TGF-β/Smad signaling pathway

Fructus Rubi (FR), a food material with medicinal value, is used in traditional Chinese medicine (TCM) for treatment of various kidney-related problems, such as impotence, spermatorrhea, and frequent urination. It is also frequently used to produce diverse functional foods in China. The purpose of this research was to assess the therapeutic effects of FR diterpene glycosides on RWPE-1 epithelial cell (RWPE-1), a human normal prostatic epithelial cell, and benign prostate hyperplasia (BPH) rats, both of which had been exposed to dihydrotestosterone (DHT) and testosterone propionate (TP), respectively, and to investigate the mechanism of action. Target proteins that could stably bind to certain diterpene glycosides were screened through drug affinity responsive target stability combined with mass spectrometry (DARTS/MS). DHT-induced RWPE-1cells were used to detect drug activity. TP was subcutaneously injected to induce BPH in rats. The extract of diterpene glycosides from FR (FDS) was orally administered for 28 days. The DHT levels in the serum and prostate tissue of the rats were measured through enzyme-linked immunosorbent assay (ELISA), and to analyze cell proliferation and epithelial-mesenchymal transition (EMT), the protein expression of prostate-specific antigen (PSA), androgen receptor (AR), steroid 5α-reductase type 2 (SRD5A2), proliferating cell nuclear antigen (PCNA), S100 calcium-binding protein A2 (S100A2), transforming growth factor-β1 (TGF-β1), E-cadherin, vimentin, and Smad4 was determined through western blotting (WB), immunohistochemistry (IHC), or immunofluorescence (IF). FDS reduced the proliferation of DHT-induced RWPE-1cells. It also significantly inhibited rat prostate enlargement; decreased DHT levels in the serum and prostate tissue; inhibited the protein expression of AR, PSA, PCNA, S100A2, TGF-β1, E-cadherin, and Smad4; and increased the protein expression of E-cadherin. The present study is the first to report that diterpene glycosides isolated from FR inhibited BPH at the cellular level, regulated the proliferation of prostate cells through the androgen signaling pathway, and prevented EMT in the prostate through the S100A2-mediated TGF-β/Smad signaling pathway. These results indicate that FDS is a promising multitarget therapy for BPH.

Picroside Ⅱ alleviates renal fibrosis through YY1-dependent transcriptional inhibition of TGFβ1

Diabetic Nephropathy (DN) has become the leading cause of end-stage renal disease worldwide. Studies have indicated that Transforming Growth Factor beta1 (TGFβ1) is the most potent factor contributing to renal fibrosis, and understanding the exact pathogenic mechanism of renal fibrosis is crucial for alleviating the condition. Previous research has identified Yin Yang 1 (YY1) as an effective inhibitor of TGF-β1. Our study, through dual-luciferase reporter gene assays and Western blot experiments, screened and obtained the small molecule compound PdⅡ. Subsequently, validation in a high-glucose-induced renal mesangial cell injury model showed that PdⅡ treatment significantly increased the expression of YY1 protein and mRNA, while correspondingly reducing the expression of TGFβ1 protein and mRNA. Dual-luciferase reporter gene assay results revealed that, compared to the control group, the luciferase transcription activity of YY1 molecules increased in the PdⅡ treatment group, and the luciferase transcription activity of TGFβ1 decreased. By further designing mutations in the binding sites between TGFβ1 and YY1 on the promoter, transfecting fluorescent enzyme reporter gene plasmids with TGFβ1 mutant promoter into mesangial cells damaged by high glucose, and then treating the cells with PdⅡ, it was observed that the luciferase transcription activity of TGFβ1 did not decrease. Therefore, these results suggest that PdⅡ may inhibit TGFβ1 transcriptional activity by activating YY1, thereby slowing down the progression of diabetic nephropathy.

Nandrolone decanoate induced kidney injury through miRNA-146a targeting IRAK1 and TRAF6 via activation of the NF-κB pathway: The effect of moderate exercise

Anabolic-androgenic steroids (AAS) abuse is linked to some abnormalities in several tissues including the kidney. However, the precise molecular mediators involved in AAS-induced kidney disorder remain elusive. The main objective of the present study was to investigate the effect of Nandrolone decanoate on kidney injury alone or in combination with moderate exercise and its related mechanisms. Thirty-two male Wistar rats were subdivided randomly into four groups. control (Con), Nandrolone (10 mg/kg)(N), Exercise (Exe), Nandrolone + Exercise (N+Exe). ResultsAfter 6 weeks, nandrolone treatment led to a significant increase in functional parameters such as serum cystatin c, urea, creatinine, albuminuria and Albumin/ creatinine ratio indicating kidney dysfunction. Moreover, nandrolone treatment increased vacuolization, focal inflammation, hemorragia, cast formation fibrosis in the renal tissue of rats. miRNA-146a increased in kidney tissue after nandrolone exposure by using RT-PCR which may be considered idealtheranomiRNAcandidates for diagnosis and treatment.Western blotting indicated that IRAK1, TRAF6, TNF-α, NF-κB, iNOS and TGF-β protein expressions were considerably elevated in the kidneys of nandrolone treated rats. Moderate exercise could alleviate the renal dysfunction, histological abnormalities and aforementioned proteins. Our findings suggested that nandrolone consumption can cause damage to kidney tissue probably through miRNA-146a targeting IRAK1 and TRAF6 via activation of the NF-κB and TGF-β pathway. These results provide future lines of research in the identification of theranoMiRNAs related to nandrolone treatment, which can be ameliorated by moderate exercise.