Protein Levels Of TGF-β1 Research Articles

Pirfenidone Attenuates Renal Tubulointerstitial Fibrosis through Inhibiting miR-21

Background: Our previous studies had shown pirfenidone (PFD) not only improved tubulointerstitial fibrosis (TIF) but also inhibited the expression of microRNA-21 (miR-21) in the renal tissue of unilateral urethral obstruction (UUO) rats. This study aims to investigate whether PFD can attenuate TIF through inhibiting miR-21 in UUO rats. Methods: Sprague Dawley rats were divided randomly into sham-operated group, UUO group, and PFD and olmesartan (Olm) treatment groups. Samples were collected on day 14. Expression of miR-21, TGF-β1, Smad3, and Smad7 mRNA in the renal tissue was detected using real-time quantitative PCR. Immunohistochemistry was performed to assess the protein expressions of collagen III, E-cadherin, and α-SMA. Automated capillary Western blotting was used to detect the quantitative expression of TGF-β1, Smad3, p-Smad3, Smad7, collagen III, E-cadherin, and α-SMA in renal tissues. The expression of miR-21 and Smad7 mRNA and the protein levels of collagen III and α-SMA were examined in the miR-21-overexpressing cell line, NRK-52E. Results: Compared with the UUO group, both PFD and Olm inhibited renal tubular dilation, diffused epithelial cell degeneration and necrosis, and reduced renal interstitial edema, inflammatory cell infiltration, and collagen fiber deposition, while no significant difference between PFD group and Olm group. Informatics-based approaches identified Smad7 as a likely candidate for regulation by miR-21. Compared with the sham group, miR-21 expression was upregulated in the UUO group resulting in the downregulation of Smad7 expression due to degradation. The overexpression of miR-21 in the in vitro model downregulated Smad7 and promoted EMT and ECM accumulation. Protein levels of TGF-β1, Smad3, p-Smad3, collagen III, and α-SMA were upregulated, while E-cadherin protein was downregulated in the UUO group than in the sham group. PFD rather than Olm decreased the expression of miR-21 and increased the expression level of Smad7 mRNA and then inhibited the TGF-β1/Smad3 signaling pathway. Olm only downregulated the TGF-β1/Smad3 signaling pathway. Conclusions: PFD improves TIF by downregulating the expression of miR-21, then elevating Smad7, and finally inhibiting the activation of the TGF-β1/Smad3 signaling pathway in UUO rats.

Effect of fresh Phragmitis Rhizoma on airway inflammation in chronic bronchitis based on TGF-β signaling pathway

This study aims to explore the mechanism of fresh Phragmitis Rhizoma against chronic bronchitis airway inflammation. The SD rats of SPF grade were divided into control group, model group, Guilongkechuanning group(GLKCN, 1.125 g·kg~(-1)), high-dose fresh Phragmitis Rhizoma group(LG-HD, 15 g·kg~(-1)), and low-dose fresh Phragmitis Rhizoma group(LG-LD, 7.5 g·kg~(-1)). The chronic bronchitis models of rats in other groups except the control group were induced by the modified smoking method. From the 15 th day of modeling, the rats were given corresponding agents by gavage for 20 consecutive days. After the last administration, the rats were sacrificed for sample collection. Enzyme-linked immunosorbent assay(ELISA) was employed to detect serum transforming growth factor-β(TGF-β) and interleukin-6(IL-6) levels. The protein expression of TGF-β, IL-1β and IL-6 in lung tissue was detected by immunohistochemical method. Masson staining was performed to detect collagen fibers and muscle fibers in lung tissue, and HE staining to detect the pathological changes of lung tissue. Human bronchial epithelial(16 HBE) cells were cultured in vitro, and CCK-8(cell counting kit-8) method was used to detect the cytotoxicity of cigarette smoke extract(CSE) and fresh Phragmitis Rhizoma. After the exposure of 16 HBE cells to 3.5% CSE and appropriate concentration(800, 400 μg·mL~(-1)) of fresh Phragmitis Rhizoma for 24 h, quantitative real-time PCR was conducted to determine the mRNA levels of TGF-β and IL-1β, and Western blot was employed to determine the protein levels of TGF-β and IL-6 in the cells. The rat model of chronic bronchitis induced by smoking was successfully established. Fresh Phragmitis Rhizoma reduced serum TGF-β and IL-6 levels, down-regulated the protein levels of TGF-β, IL-1β, and IL-6 in lung tissue, and alleviated pathological changes and fibrotic lesions in lung tissue. Moreover, it down-regulated the CSE-induced protein expression of TGF-β and IL-6 as well as the mRNA level of TGF-β in 16 HBE cells. These results indicated that fresh Phragmitis Rhizoma could prevent airway inflammation from chronic bronchitis and promote cell repair by inhibiting the TGF-β signaling pathway.

Exploring the Effect of Jiawei Buguzhi Pills on TGF-β-Smad Pathway in Postmenopausal Osteoporosis Based on Integrated Pharmacological Strategy.

Objective To explore the effect of Jiawei Buguzhi Pills (JWBGZP) on the TGF-β-Smad pathway in postmenopausal osteoporosis (PMO) based on integrated pharmacological strategy. Method The ETCM database was used to collect JWBGZP. GeneCards and OMIM databases were utilized to obtain PMO-related genes. Cytoscape was used for network construction and analysis, and DAVID was used for GO and KEGG enrichment analysis of key targets. Animal experiments and cell experiments were conducted to further explore the mechanism. The bone mass density was detected by dual-energy X-ray bone densitometer. The TGF-β1 and Smad4 mRNA in bone tissue were detected by RT-qPCR. The TGF-β1 and Smad4 protein in bone tissue were detected by the western blot. The TGF-β1 and Smad4 protein in osteoblasts were determined by immunohistochemistry. Result A total of 721 JWBGZP potential targets and 385 PMO-related genes were obtained. The enrichment analysis showed that JWBGZP may regulate the TGF-beta signaling pathway, oxidation-reduction process, aging, response to hypoxia, response to ethanol, negative regulation of cell proliferation, PI3K-Akt, HIF-1, and other signaling pathways. The animal experiments showed that compared with the model group, the femoral bone mineral density and lumbar bone mineral density of the JWBGZP group increased (P < 0.05); the expression levels of TGF-β1 and Smad mRNA and proteins in the JWBGZP group were significantly higher (P < 0.05). The cell experiment results showed a large number of osteoblast stained blue-purple and orange-red calcified nodules. The expression levels of TGF-β1 and Smad proteins in the JWBGZP group were significantly higher than those in the blank control group and the sham operation group, and the protein expression levels in the model group were the lowest (P < 0.05). Conclusion JWBGZP may be involved in PI3K-Akt, HIF-1, estrogen, prolactin, and other signaling pathways and regulate MAPK1, AKT1, PIK3CA, JAK2, and other gene targets, regulate bone metabolism, and thereby treat PMO.

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.

HIV Tat Protein Induces Myocardial Fibrosis Through TGF-β1-CTGF Signaling Cascade: A Potential Mechanism of HIV Infection-Related Cardiac Manifestations.

Human immunodeficiency virus (HIV) infection is a risk factor of cardiovascular diseases (CVDs). HIV-infected patients exhibit cardiac dysfunction coupled with cardiac fibrosis. However, the reason why HIV could induce cardiac fibrosis remains largely unexplored. HIV-1 trans-activator of transcription (Tat) protein is a regulatory protein, which plays a critical role in the pathogenesis of various HIV-related complications. In the present study, recombinant Tat was administered to mouse myocardium or neonatal mouse cardiac fibroblasts in different doses. Hematoxylin-eosin and Masson's trichrome staining were performed to observe the histological changes of mice myocardial tissues. EdU staining and MTS assay were used to evaluate the proliferation and viability of neonatal mouse cardiac fibroblasts, respectively. Real-time PCR and western blot analysis were used to detect CTGF, TGF-β1, and collagen I mRNA and protein expression levels, respectively. The results showed that Tat promoted the occurrence of myocardial fibrosis in mice. Also, we found that Tat increased the proliferative ability and the viability of neonatal mouse cardiac fibroblasts. The protein and mRNA expression levels of TGF-β1 and CTGF were significantly upregulated both in Tat-treated mouse myocardium and neonatal mouse cardiac fibroblasts. However, co-administration of TGF-β inhibitor abrogated the enhanced expression of collagen I induced by Tat in neonatal mouse cardiac fibroblasts. In conclusion, Tat contributes to HIV-related cardiac fibrosis through enhanced TGF-β1-CTGF signaling cascade.

Scutellarin-induced A549 cell apoptosis depends on activation of the transforming growth factor-β1/smad2/ROS/caspase-3 pathway.

Scutellarin plays an anti-tumor role in A549 lung cancer cells, but the underlying mechanism is unclear. In this study, scutellarin was used to treat A549 cells for 12, 24, and 48 h, followed by the addition of Tempo, a selective scavenger of mitochondrial reactive oxygen species (ROS) and SB431542, a transforming growth factor (TGF)-β1 receptor inhibitor. A dihydroethidium fluorescence probe was used to measure the intracellular ROS level, Cell Counting Kit-8 (CCK-8) was used to detect cell viability, and flow cytometry was performed to examine apoptosis. Western blots were used to detect the total protein level of TGF-β1, p-smad2, and cleaved caspase-3 in A549 cells. The results showed that scutellarin significantly inhibited cell viability and increased apoptosis. Scutellarin also promoted intracellular ROS production, TGF-β1/smad2 signaling pathway activation, and cleaved caspase-3 expression, which was partly reversed by Tempo. Moreover, scutellarin-induced intracellular ROS production and cleaved caspase-3 expression were inhibited by blocking the TGF-β1/smad2 pathway with SB431542. In conclusion, scutellarin promoted apoptosis and intracellular ROS accumulation, which could be abrogated by Tempo and SB431542 treatment in A549 cells. Our study indicated that scutellarin induced A549 cell apoptosis via the TGF-β1/smad2/ROS/caspase-3 pathway.

Effects of Sitagliptin on myocardial remodeling and autophagy in diabetic mice and its mechanism

Objective: To investigate the effects of Sitagliptin on myocardial remodeling and autophagy in diabetic mice and its possible mechanisms. Methods: C57 mice aged ten weeks were treated with streptozocin (STZ) at the dose of 50 mg/(kg·d) by intraperitoneal injections for five consecutive days, and the level of fasting blood glucose concentration was higher than 16.7 mmol/l after seven days indicated that the diabetic model was established successfully. Mice were divided into four groups, including control group (n=10) which was intraperitoneally injected with the same volum of saline, the model group (n=8), Sitagliptin treatment group(diabetic mice were treated with Sitagliptin at the dose of 10 mg/(kg·d)by gavage, n=8) and the inhibitor group(diabetic mice were treated with Compound C, an AMPK inhibitor, at the dose of 10 mg/(kg·d) by intraperitoneal injection, n=8). After six weeks, all the mices were weighted and then put to death and the hearts were separated to caculate ventricular /body weight ratio. Hemaloxylin-Eosin (HE) staining was used to observe the cell morphology and masson staining was used to observe interstitial fibrosis. Western blot was used to test the heart protein expressions of Connexin43(Cx43), adenosine 5'-monophosphate -activated protein kinase (AMPK), brain natriuretic peptide(BNP), transforming growth factor(TGF-β) and LC3B. Results: After six weeks of treatment, compared with control group, the ventricular /body weight ratio was improved (P＜0.05), The cardiomyocyte hypertrophy and increased fibrosis were observed in the model group. The expression levels of BNP and TGF-β were increased, while the expression levels of Cx43,LC3B and AMPK were decreased significantly(P＜0.05). However, compared with model group, treatment with Sitagliptin decreased BNP, TGF-β protein levels and increased Cx43 and LC3B protein levels, while Compound C could inhibit the upregulation of Cx43, LC3B and AMPK protein (P＜0.05). Conclusion: Sitagliptin could improve cardiac hypertrophy and decrease interstitial fibrosis and AMPK-related signaling pathways was involved in the regulation of Cx43 and autophagy.

Hirsutella sinensis fungus improves cardiac function in mouse model of heart failure

Cordyceps sinensis, including Hirsutella sinensis, is a highly valuable traditional Chinese medicine and is used to treat patients with pulmonary heart disease in clinical practice. However, the underlying mechanisms of its effects remain unclear. In this study, a mouse model of heart failure established by non-thoracic, transverse aortic constriction (TAC) was developed to determine the underlying mechanisms of therapeutic effects of Hirsutella sinensis fungus (HSF) powder. The results showed that HSF treatment remarkably ameliorated myocardial hypertrophy, collagen fiber hyperplasia, and cardiac function in mice with heart failure. Using transcriptional and epigenetic analyses, we found that the mechanism of HSF mainly involved a variety of signaling pathways related to myocardial fibrosis and determined that HSF could reduce the levels of TGF-β1 proteins in heart tissue, as well as type I and III collagen levels. These data suggest that HSF alleviates heart failure, inhibits irreversible ventricular remodeling, and improves cardiac function through the regulation of myocardial fibrosis-related signaling pathways, which can provide novel opportunities to improve heart failure therapy.

Astaxanthin Ameliorates high-fat diet-induced cardiac damage and fibrosis by upregulating and activating SIRT1.

This study evaluated the protective effect of astaxanthin (ASX) against high-fat diet (HFD)-induced cardiac damage and fibrosis in rats and examined if the mechanism of protection involves modulating SIRT1. Rat were divided into 5 groups (n = 10/group) as: 1) control: fed normal diet (3.82 kcal/g), 2) control + ASX (200 mg/kg/orally), 3) HFD: fed HFD (4.7 kcal/g), 4) HFD + ASX (200 mg/kg/orally), and HFD + ASX + EX-527 (1 mg/kg/i.p) (a selective SIRT1 inhibitor). All treatments were conducted for 14 weeks. Administration of ASX reduced cardiomyocyte damage, inhibited inflammatory cell infiltration, preserved cardiac fibers structure, prevented collagen deposition and protein levels of TGF-β 1 in the left ventricles (LVs) of HFD-fed rats. In the LVs of both the control and HFD-fed rat, ASX significantly reduced levels of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and p-smad2/3 (Lys19) but increased the levels of glutathione (GSH), catalase, and manganese superoxide dismutase (MnSOD). Concomitantly, it increased the nuclear activity of Nrf2 and reduced that of NF-κB p65. Furthermore, administration of ASX to both the control and HFD-fed rats increased total and nuclear levels of SIRT1, stimulated the nuclear activity of SIRT1, and reduced the acetylation of Nrf2, NF-κB p65, and Smad3. All these cardiac beneficial effects of ASX in the HFD-fed rats were abolished by co-administration of EX-527. In conclusion, ASX stimulates antioxidants and inhibits markers of inflammation under basal and HFD conditions. The mechanism of protection involves, at least, activation SIRT1 signaling.

Antifibrotic Effects of (-)-Epicatechin on High Glucose Stimulated Cardiac Fibroblasts.

Cardiac fibrosis is one of the hallmarks of a diabetic cardiomyopathy. When activated, cardiac fibroblasts (CFs) increase the production of extracellular matrix proteins. Transforming growth factor (TGF)-β1 is known to mediate cardiac fibrosis through the SMAD pathway. High glucose (HG = 25 mM) cell culture media can activate CFs using TGF-β1. There is a need to identify effective antifibrotic agents. Studies in animals indicate that treatment with (-)-epicatechin (Epi) appears capable of reducing myocardial fibrosis. Epi binds to G-protein coupled estrogen receptor (GPER) and activates downstream pathways. We evaluated the potential of Epi to mitigate the development of a profibrotic phenotype in HG stimulated CFs. CF primary cultures were isolated from young male rats and were exposed for up to 48 h HG media and treated with vehicle or 1 μM Epi. Relevant profibrotic end points were measured by the use of various biochemical assays. HG exposure of CFs increased TGF-β1 protein levels by ∼15%, fibronectin ∼25%, urea levels ∼60%, proline incorporation ∼70%, and total collagen ∼15%. Epi treatment was able to significantly block HG induced increases in TGF-β1, fibronectin, urea, proline, and total collagen protein levels. GPER levels were reduced by HG and restored in CFs treated with Epi an effect associated with the activation (i.e., phosphorylation) of c-Src. Epi treatment also reverted SMAD levels. Altogether, results demonstrate that CFs cultured in HG acquire a profibrotic phenotype, which is blocked by Epi an effect, likely mediated at least, in part, by GPER effects on the SMAD/TGF-β1 pathway.

Promoting effect of pomegranate peel extract on second-degree burn wound-healing through VEGF-A and TGF-β1 regulation

Pomegranate peel extract (PPE), a polyphenolic compound derived from pomegranate, has been widely concerned for its anti-oxidant, anti-inflammatory, and bacteriostatic effects. The potential therapeutic effect of PPE on burn injury was investigated, and its possible mechanisms were explored. Minipigs with second-degree burn were treated with PPE, Jing Wan Hong, and silver sulfadiazine. Hematoxylin–eosin (HE) staining was performed to detect burn severity, and then biological tissues were biopsied on days 0, 7, 14, 21, and 28 after administration. Immunohistochemistry, western blot, and real-time polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression levels of VEGF-A and TGF-β1 in skin tissues after treatment with PPE. Furthermore, the skin wound healing at different time points was monitored by macroscopic observation. HE showed that after 28-day PPE treatment, the morphology of the skin tissue showed a significant improvement. Macroscopic data monitoring indicated that the decrustation and fur growing time was shortened. Meanwhile, the rate of wound healing increased after PPE treatment. The combination of immunohistochemistry, western blotting, and RT-PCR showed that after PPE treatment, expression of VEGF-A and TGF-β1 increased sharply on day 7, maintaining a high level until day 14, showing a downward trend on day 21, and approaching normal levels on day 28. However, in the model group, the protein and mRNA expression levels of VEGF-A and TGF-β1 increased on day 28 after burn injury, which was a slow process. Results indicated that compared with the model group, the peak expression level of VEGF-A and TGF-β1 was earlier, which was consistent with decrustation, shortening of fur growing time, and improvement of wound healing rate in minipig second-degree burn model. PPE showed a significant promoting effect on minipig second-degree burn model, which might be associated with the upregulation of the protein and gene expression levels of VEGF-A and TGF-β1.

RETRACTED ARTICLE: Taurine ameliorates thioacetamide induced liver fibrosis in rats via modulation of toll like receptor 4/nuclear factor kappa B signaling pathway

Liver fibrosis is a significant health problem that can cause serious illness and death. Unfortunately, a standard treatment for liver fibrosis has not been approved yet due to its complicated pathogenesis. The current study aimed at assessing the anti-fibrotic effect of taurine against thioacetamide induced liver fibrosis in rats through the modulation of toll like receptor 4/nuclear factor kappa B signaling pathway. Both concomitant and late taurine treatment (100 mg/kg, IP, daily) significantly reduced the rise in serum ALT and AST activities and significantly reversed the decrease in serum albumin and total protein. These results were confirmed by histopathological examinations and immunehistochemical inspection of α-SMA, caspase-3 and NF-κB. The antioxidant potential of taurine was verified by a marked increase of GSH content and a reduction of MDA level in liver tissue. The anti-fibrotic effects of taurine were evaluated by investigating the expression of TLR4, NF-κB. The protein levels of IL-6, LPS, MyD88, MD2, CD14, TGF-β1 and TNF-α were determined. Docking studies were carried out to understand how taurine interacts inside TLR4-MD2 complex and it showed good binding with the hydrophobic binding site of MD2. We concluded that the anti-fibrotic effect of taurine was attributable to the modulation of the TLR4/NF-κB signaling.

Calycosin inhibits breast cancer cell migration and invasion by suppressing EMT via BATF/TGF-β1.

In this study, we investigated the effects of calycosin on breast cancer cell progression and their underlying mechanisms. Calycosin dose- and time-dependently inhibited proliferation, migration, and invasion by T47D and MCF-7 breast cancer cells by downregulating basic leucine zipper ATF-like transcription factor (BATF) expression. Moreover, BATF promoted breast cancer cell migration and invasiveness by increasing TGFβ1 mRNA and protein levels. Bioinformatics analysis, dual luciferase reporter assays, and chromatin immunoprecipitation assays confirmed the presence of BATF-binding sites in the promoter sequence of TGFβ1 gene. Calycosin treatment inhibited epithelial-mesenchymal transition (EMT) of breast cancer cells by significantly increasing E-cadherin levels and decreasing N-cadherin, Vimentin, CD147, MMP-2, and MMP-9 levels through downregulation of BATF and TGFβ1. TGFβ1 knockdown reduced the migration and invasiveness of BATF-overexpressing breast cancer cells, whereas incubation with TGFβ1 enhanced the migration and invasiveness of calycosin-treated breast cancer cells. Our findings demonstrated that calycosin inhibited EMT and progression of breast cancer cells by suppressing BATF/TGFβ1 signaling. This suggests calycosin would be a promising therapeutic option for breast cancer patients.

Expression of Transforming Growth Factor β1, Smad3, and Phospho-Smad3 in Somatotropinomas and Their Relationship to Tumor Behavior

To investigate the role of transforming growth factor β1 (TGF-β1), Smad3, and phospho-Smad3 (p-Smad3) in the invasion of somatotropinomas. In total, 45 somatotropinomas were obtained from patients who underwent surgery for the first time between 2011 and 2015 at Beijing Tiantan Hospital. The expression of TGF-β1, Smad3, and p-Smad3 was examined by western blot, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry in somatotropinomas, and factors correlated with tumor invasion were analyzed. A total of 13 invasive somatotropinomas and 32 noninvasive somatotropinomas were enrolled in the study. TGF-β1 protein (P < 0.01) and mRNA (P < 0.01) levels were significantly less in the invasive somatotropinomas than noninvasive somatotropinomas. There was no significant difference in Smad3 protein level or Smad3 mRNA level between invasive somatotropinomas and noninvasive somatotropinomas. However, the p-Smad3 protein level was significantly less in the invasive somatotropinomas than noninvasive somatotropinomas (P < 0.01). Univariate analysis demonstrated that TGF-β1 (P < 0.01) and p-Smad3 scores (P < 0.01) were associated with invasion. In multivariate analysis, p-Smad3 scores remained a significantly independent predictor of invasion (odds ratio 0.897, 95% confidence interval 0.834-0.964, P < 0.05). Low expression of p-Smad3 is correlated with invasion of somatotropinomas.

MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY

Abstract Background and Aims Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4). Conclusion DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.

Pilose Antler Peptide-3.2KD Ameliorates Adriamycin-Induced Myocardial Injury Through TGF-β/SMAD Signaling Pathway.

Adriamycin (ADR)-based combination chemotherapy is the standard treatment for some patients with tumors in clinical, however, long-term application can cause dose-dependent cardiotoxicity. Pilose Antler, as a traditional Chinese medicine, first appeared in the Han Dynasty and has been used to treat heart disease for nearly a thousand years. Previous data revealed pilose antler polypeptide (PAP, 3.2KD) was one of its main active components with multiple biological activities for cardiomyopathy. PAP-3.2KD exerts protective effects againt myocardial fibrosis. The present study demonstrated the protective mechanism of PAP-3.2KD against Adriamycin (ADR)-induced myocardial injury through using animal model with ADR-induced myocardial injury. PAP-3.2KD markedly improved the weight increase and decreased the HW/BW index, heart rate, and ST height in ADR-induced groups. Additionally, PAP-3.2KD reversed histopathological changes (such as disordered muscle bundles, myocardial fibrosis and diffuse myocardial cellular edema) and scores of the heart tissue, ameliorated the myocardial fibrosis and collagen volume fraction through pathological examination, significantly increased the protein level of Bcl-2, and decreased the expression levels of Bax and caspase-3 in myocardial tissue by ELISA, compared to those in ADR-induced group. Furthermore, ADR stimulation induced the increased protein levels of TGF-β1 and SMAD2/3/4, the increased phosphorylation levels of SMAD2/3 and the reduced protein levels of SMAD7. The expression levels of protein above in ADR-induced group were remarkably reversed in PAP-3.2KD-treated groups. PAP-3.2KD ameliorated ADR-induced myocardial injury by regulating the TGF-β/SMAD signaling pathway. Thus, these results provide a strong rationale for the protective effects of PAP against ADR-induced myocardial injury, when ADR is used to treat cancer.

Effects of Growth Hormone on Pancreatic Cancer Derived Exosomes

In 2020, the National Cancer Institute (NCI) estimates 57,600 new cases and 47,050 deaths in the US due to pancreatic ductal adenocarcinoma (PDAC). A dismal 10% five-year overall survival rate in PDAC is attributed to late diagnosis, limited treatment options, a remarkably high metastasis rate, and resistance of this cancer to available therapies. Therefore, a better understanding of the mechanisms of how PDAC tumors acquire drug resistance and spread to distal parts of the body are necessary for developing novel therapeutic approaches. Exosomes, microscopic vesicles released from most cells (both tumor and non-tumor) have been recently established to play a significant role in cell to cell communication. Exosomes modulate their target cell responses systematically depending on the nature of exosomal cargoes (nucleic acids, proteins, and lipids). PDAC derived exosomes have been implicated to promote metastasis via forming a pre-metastatic niche of cells as well as enhancing drug resistance. Growth hormone (GH) secreted primarily by the pituitary gland promotes metastasis and drug resistance as shown by plethora of studies. No study has directly assessed the effect of GH on exosomal cargoes in terms of promoting metastases and drug resistance. In this report, we show that GH modulates various pancreatic cancer cell exosomal cargoes which in turn potentially amplifies tumor invasion and metastases. Our data shows that GH treatment on human and mouse PDAC cells increases the exosomal protein levels of TGFβ - a critical inducer of epithelial-to-mesenchymal transition (EMT, a process leading to metastasis). In addition, GH treatment also increases extracellular matrix-degrading enzymes, MMP2 and 9, as well as multi-drug efflux pump ABCC1, ABCB1, and ABCG2 in PDAC cells. These results strongly implicate GH action in driving EMT and chemoresistance via exosomes in pancreatic cancer. Exosomes have a crucial impact especially in the areas of diagnostics and therapeutics. This report is the first to show that GH modulates the effects of exosomes secreted by pancreatic cancer cells. Acknowledgement: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Student Enhancement Award and Edison Biotechnology Institute.

Combination of Niacinamide and Undenatured Collagen Type II Modulates Inflammatory Response in Monosodium Iodoacetate‐Induced Osteoarthritis in Rats

The present work was to investigate the effects of oral supplementation of chondroprotective influence of niacinamide (NA) and undenatured type II collagen (UC-II®) on the inflammation and the joint pain behavior of rats in a monoiodoacetate (MIA)-induced experimental osteoarthritis (OA) model. Forty-nine Wistar rats were allocated into seven groups, namely; 1) control (no MIA); 2) MIA, positive control as 1 mg MIA-induced knee osteoarthritis; 3) MIA+UCII, MIA + UCII at 0.66 mg/kg/BW; 4) MIA+NA40, MIA + NA at 40 mg/kg BW; 5) MIA+NA200, MIA (1 mg) + NA at 200 mg/kg BW; 6) MIA+UCII+NA40 : MIA (1 mg) + UCII at 0.66 mg +NA at 40 mg/kg BW and 7) MIA+UCII+NA200: MIA (1 mg) + UCII at 0.66 mg + NA at 200mg/kg BW. OA outcomes such as gait test (paw area, paw width, stride length) of the knee joint was analyzed. Serum interleukin 1beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-alpha (TNF-α), cartilage oligomeric matrix protein (COMP), C-reactive protein (CRP), serum malondialdehyde (MDA) and knee joint matrix metalloproteinase-3 (MMP-3), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ), and transforming growth factor-beta (TGF-β)-1 levels were analyzed. Analysis of variance (ANOVA) test and post-hoc Tukey test was used for multiple comparisons of the groups. Serum IL-1β, IL-6, TNF-α, COMP, and CRP decreased in rats supplemented with UCII and in combinations with NA compared with control rats (p < 0.05). Rats supplemented with UCII and in combinations with NA reduced serum MDA and knee joint MMP-3, NF-κβ, and TGF-β protein levels (p < 0.05). The UCII supplementation to OA rats decreased the Kellgren-Lawrence and the Mankin scores (p < 0.05) indicating a reduced in OA disease severity. These results suggest undenatured type II collagen combined with niacinamide is effective in reducing OA outcomes.

Corilagin alleviates hypertrophic scars via inhibiting the transforming growth factor (TGF)-β/Smad signal pathway

AimsExploring the effects of corilagin on hypertrophic scar (HS) and its underlying mechanisms. Main methodsHuman HS-derived fibroblasts (HSFs) were isolated and treated with corilagin. To investigate the effects of corilagin on HSFs, quantitative real time polymerase chain reaction (qRT-PCR), western blotting, wound healing, and immunofluorescence assays were performed. These effects were confirmed in a rabbit ear scar model by histological and immunohistochemical studies. Lastly, western blot assay was performed to detect the protein levels of several components of the transforming growth factor (TGF)-β/Smad signaling pathway, as well as the protein levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs). Key findingsCorilagin showed multiple effects on HSFs, including does-dependent inhibition of collagen production, cell proliferation, and migration, besides suppression of the activation of HSFs. Moreover, corilagin suppressed HS formation and collagen deposition in a rabbit ear scar model. Corilagin also inhibited fibroblast proliferation and α-smooth muscle actin (α-SMA) expression in vivo. Finally, western blot analysis revealed that corilagin downregulated the protein levels of TGF-β1 and TGF-β receptor type I (TGFβRI), thus lowering the level of p-smad2/3, also affected the protein levels of MMPs and TIMP1. SignificanceCorilagin could be a potential agent for HS treatment through the inhibition of extracellular matrix (ECM) deposition and multiple functions of fibroblasts.

NLRP3-inflammasome activation is associated with epithelial-mesenchymal transition and progression of colorectal cancer.

Objective(s):Since activation of NLRP3 inflammasome results in the production of interleukin-1β (IL 1β) and initiation of inflammation as the key players in development of cancer, this study investigated possible activation of NLRP3 inflammasome during the progression of colorectal cancer (CRC) and evaluated the role of NLRP3 inflammasome in epithelial-mesenchymal transition (EMT) process. Materials and Methods:Tissue samples were collected from cancerous (test) and adjacent normal tissues (control) of forty-three male CRC patients (18 grade I and 25 grade III). The gene expression and protein levels were determined by qRT PCR and Western blotting, respectively, and tissue morphological was examined by histopathology. Results:The gene and protein expression levels of transforming growth factor-β (TGF β), IL 1β, nuclear factor κB (NF κB), NLRP3, and caspase-1, as well as the enzyme activity of caspase-1, were significantly increased in CRC. mRNA and protein levels of TGF-β, mature IL 1β, NF κB, and NLRP3 were higher in patients with grade III. EMT markers N cadherin, vimentin, and MMP 9 markedly increased in CRC, and were higher in grade III than grade I, whereas expression of E-cadherin declined by the progression of CRC. NLRP3 protein level was inversely correlated with E-cadherin whereas it positively was correlated with IL 1β, active NF κB, N cadherin, vimentin, and MMP 9. Conclusion:This study for the first time showed that activation of NLRP3 inflammasome contributed to the progression of CRC and is correlated with the EMT process. Although the present study showed that EMT markers are positively correlated with tumor grade, further investigations are required to strongly link the EMT markers to the progression of CRC.