The mechanism of Shenlong Jianji treatment of idiopathic pulmonary fibrosis inhibits fibroblast-to-myofibroblast transformation via the TGF-ß1/smads signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlong Jianji (SLJJ) is a Chinese herbal compound composed of traditional medicines for supplementing Qi, nourishing Yin, promoting blood circulation, and removing obstruction in channels. It is widely used to treat idiopathic pulmonary fibrosis (IPF) in China. However, the underlying mechanism of SLJJ remains unclear. AIM OF THIS STUDY: To elucidate the efficacy and mechanisms of SLJJ in the treatment of IPF through in vivo and in vitro experiments. MATERIAL AND METHODS: 84 Wistar rats were randomly and equally divided into 7 groups: the control group (CTRL), the sham operation group (SHAM), the model group (IPF), the low dose of SLJJ group (L-SLJJ), the middle dose of SLJJ group (M-SLJJ), the high dose of SLJJ group (H-SLJJ), and the pirfenidone group (PFD). The rats in the CTRL, SHAM, and IPF groups were given normal saline each time for 28 days; the SLJJ groups were given Shenlong Jianji (9 g kg-1·d-1, 18 g kg-1·d-1, 36 g kg-1·d-1), and pirfenidone was administered as a sequential dose. After 28 days, the general condition of the rats was evaluated, and samples were collected. The lung coefficient was measured. The pathological changes of lung in each group were observed by H&E staining and Masson staining. α-SMA, collagen 1, and E-cadherin proteins were detected by immunohistochemistry. α-SMA, collagen 1, vimentin, E-cadherin, N-cadherin, TGF-ß1, smad2, and smad3 proteins were detected by WB in vivo.In vitro, A scratch test was used to assess the ratio of cell migration. α-SMA, vimentin, E-cadherin, and N-cadherin protein levels were evaluated by a cellular immunofluorescence assay. TGF-ß1/smads signaling pathway was detected by WB. HPLC-Q-TOF/MS analysis was used to identify the active compounds in the SLJJ. Molecular docking determined the free binding energy of the compound with the TGF-ß1 protein. RESULTS: SLJJ significantly improved the respiratory symptoms, heart rate, mental state, and food intake of IPF group rats and decreased the lung coefficient. In the IPF group, inflammatory cells were infiltrated, and the thickened alveoli wall and alveoli collapse were shown, while significantly alleviating pathological changes in the SLJJ and PFD groups. Masson staining showed that SLJJ and PFD decreased the collagen expression. Immunohistochemical results showed that the expressions of α-SMA, collagen 1, and N-cadherin decreased in the SLJJ and PFD groups, while E-cadherin increased significantly compared with the IPF group. SLJJ regulates TGF-ß1/smads signaling pathway proteins in vivo. SLJJ decreased the ratio of migration in HFL-1 cells; SLJJ reduced the fluorescence intensity of α-SMA, vimentin, and N-cadherin and increased the fluorescence intensity of E-cadherin in primary rat lung (PRL) fibroblast cells and HFL-1 cells. WB results showed that SLJJ significantly down-regulated α-SMA, Vimentin, N-cadherin, TGF-ß1, smad2, and p-smad2/3 proteins expression and up-regulated E-cadherin protein expression in vitro, whereas SRI-011381 (a TGF-ß1 agonist) antagonized the effects of SLJJ. CONCLUSION: SLJJ inhibits idiopathic pulmonary fibrosis. The TGF- ß1/Smads signaling pathway can be the target of SLJJ, which inhibits fibroblast-to-myofibroblast transformation and is expected to be a new drug for the treatment of IPF.

Total sesquiterpenoids from Eupatorium lindleyanum DC. attenuate bleomycin-induced lung fibrosis by suppressing myofibroblast transition.

Eupatorium lindleyanum DC. has been used as a functional food in China for a long time. However, the antifibrotic activity of total sesquiterpenoids from Eupatorium lindleyanum DC. (TS-EL) is still unknown. In this study, we discovered that TS-EL reduced the increase in α-smooth muscle actin (α-SMA), type I collagen and fibronectin content, the formation of cell filaments and collagen gel contraction in transforming growth factor-ß1-stimulated human lung fibroblasts. Intriguingly, TS-EL did not change the phosphorylation of Smad2/3 and Erk1/2. TS-EL decreased the levels of serum response factor (SRF), a critical transcription factor of α-SMA, and SRF knockdown alleviated the transition of lung myofibroblasts. Furthermore, TS-EL significantly attenuated bleomycin (BLM)-induced lung pathology and collagen deposition and reduced the levels of two profibrotic markers, total lung hydroxyproline and α-SMA. TS-EL also decreased the levels of SRF protein expression in BLM-induced mice. These results suggested that TS-EL attenuates pulmonary fibrosis by inhibiting myofibroblast transition via the downregulation of SRF.

Glutamine uptake and catabolism is required for myofibroblast formation and persistence.

BACKGROUND: Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. METHODS: Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1-/-) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. RESULTS: Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFß-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. CONCLUSIONS: These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.

Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation.

Radiofrequency ablation (RFA) of intrahepatic tumors induces distant tumor growth through activation of interleukin 6/signal transducer and activator of transcription 3 (STAT3)/hepatocyte growth factor (HGF)/tyrosine-protein kinase Met (c-MET) pathway. Yet, the predominant cellular source still needs to be identified as specific roles of the many types of periablational infiltrating immune cells requires further clarification. Here we report the key role of activated myofibroblasts in RFA-induced tumorigenesis and successful pharmacologic blockade. Murine models simulating RF tumorigenic effects on a macrometastatic tumor and intrahepatic micrometastatic deposits after liver ablation and a macrometastatic tumor after kidney ablation were used. Immune assays of ablated normal parenchyma demonstrated significantly increased numbers of activated myofibroblasts in the periablational rim, as well as increased HGF levels, recruitment other cellular infiltrates; macrophages, dendritic cells and natural killer cells, HGF dependent growth factors; fibroblast growth factor-19 (FGF-19) and receptor of Vascular Endothelial Growth Factor-1 (VEGFR-1), and proliferative indices; Ki-67 and CD34 for microvascular density. Furthermore, macrometastatic models demonstrated accelerated distant tumor growth at 7d post-RFA while micrometastatic models demonstrated increased intrahepatic deposit size and number at 14 and 21 days post-RFA. Multi-day atorvastatin, a selective fibroblast inhibitor, inhibited RFA-induced HGF and downstream growth factors, cellular markers and proliferative indices. Specifically, atorvastatin treatment reduced cellular and proliferative indices to baseline levels in the micrometastatic models, however only partially in macrometastatic models. Furthermore, adjuvant atorvastatin completely inhibited accelerated growth of macrometastasis and negated increased micrometastatic intrahepatic burden. Thus, activated myofibroblasts drive RF-induced tumorigenesis at a cellular level via induction of the HGF/c-MET/STAT3 axis, and can be successfully pharmacologically suppressed.

[Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts].

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Shenweifang-containing serum inhibits transforming growth factor-ß1 induced myofibroblast differentiation in normal rat kidney interstitial fibroblast cell.

OBJECTIVE: To investigate the efficacy of Shenweifang (SWF)-containing serum on transforming growth factor (TGF)-ß1-induced fibroblast-myofibroblast transition in normal rat kidney interstitial fibroblast cells (NRK-49F). METHODS: Sprague-Dawley rats were gavaged with one of five solutions: (a) saline; (b) saline plus low-dose SWF; (c) saline plus medium-dose SWF; (d) saline plus highdose SWF; and (e) saline plus valsartan. NRK-49F cells were treated with TGF-ß1 and cultured using serum from the gavaged rats. RESULTS: TGF-ß1 treatment increased the expression of α-smooth muscle actin, proliferating cell nuclear antigen, collagen I, Smad3, mitogen-activated protein kinase (MAPK) 10, and c-Jun N-terminal kinase (JNK) 3 and induced abnormalities in cell morphology, cell cycle progression, and cell proliferation. CONCLUSIONS: SWF- or valsartan-containing serum corrected (or partially corrected) TGF-ß1-induced abnormal changes in this in vitro system. SWF-containing serum reversed abnormalities in morphology, cell cycle progression, and proliferation in TGF-ß1-treated NRK49F cells, probably by blocking the TGF-ß1/Smads and TGF-ß1/MAPK/JNK pathways.

Glycyrrhiza uralensis root extract ameliorates high glucose-induced renal proximal tubular fibrosis by attenuating tubular epithelial-myofibroblast transdifferentiation by targeting TGF-ß1/Smad/Stat3 pathway.

Growing evidences indicate that high glucose toxicity-associated fibrotic effects play a pivotal role in diabetic nephropathy (DN). Tubular epithelial-myofibroblast transdifferentiation is a major hallmark of renal fibrosis event under diabetic stress. Roots of Glycyrrhiza uralensis (Radix glycyrrhizae) used as a sweetener and traditional Chinese medicine possess high potential for renal protection. In this study, a cell model for high glucose (HG) injury with HK-2 renal proximal tubular epithelial cell line and a type-II-diabetes model with Apoeem1/Narl /Narl mice was established and the beneficial effects of aqueous R. glycyrrhizae extract (RGE) was investigated. RGE-induced regulation on the high glucose-induced excessive production of TGF-ß1 and the Smad/Stat3 mechanisms of renal fibrosis were determined. HK-2 cells were challenged with 45 mM of high glucose for 48 hr. Following high glucose challenge, the cells were treated with 0.5, 1, and 1.5 mg/ml concentrations of RGE. The effect of RGE on DN was determined using high fructose diet-induced type-II-diabetes in Apoeem1/Narl /Narl mice models. Our results showed that RGE suppressed the expression of HG-induced TGFß signaling and associated fibrosis mechanism better than the pharmacological drug acarbose. These data suggest that RGE as a potential herbal supplement in attenuating fibrosis-associated diabetic nephropathy and a potential agent in diabetes treatments.

Tetramethylpyrazine Retards the Progression and Fibrogenesis of Endometriosis.

The development of more efficacious, non-hormonal therapeutics for endometriosis is still an unmet medical need begging to be fulfilled. Growing evidence indicates that endometriotic lesions are wounds undergoing repeated tissue injury and repair, and, as such, platelets play an important role in lesional progression. Tetramethylpyrazine (TMP), a compound derived from a herb that has been used for thousands of years to combat "blood stasis" in traditional Chinese medicine, is a prescription drug in China for the treatment of cerebrovascular disorders. We tested the hypothesis that TMP can decelerate lesional progression through arresting epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), and fibrogenesis. We found in our in vitro experiments that TMP treatment suppresses platelet-induced EMT, FMT, cellular contractility, and collagen production in a concentration-dependent manner. We also showed that in a mouse model of endometriosis, treatment with TMP significantly reduced lesion weight and the extent of lesional fibrosis and improved hyperalgesia, mostly likely through the reduction of lesional aggregation of platelets and the lesional expression of markers of EMT, FMT, and fibrogenesis. In light of our results and in view of its excellent safety profiles, TMP appears to be a promising drug candidate for treating endometriosis.

IL-15 Prevents Renal Fibrosis by Inhibiting Collagen Synthesis: A New Pathway in Chronic Kidney Disease?

Chronic kidney disease (CKD), secondary to renal fibrogenesis, is a public health burden. The activation of interstitial myofibroblasts and excessive production of extracellular matrix (ECM) proteins are major events leading to end-stage kidney disease. Recently, interleukin-15 (IL-15) has been implicated in fibrosis protection in several organs, with little evidence in the kidney. Since endogenous IL-15 expression decreased in nephrectomized human allografts evolving toward fibrosis and kidneys in the unilateral ureteral obstruction (UUO) model, we explored IL-15's renoprotective role by pharmologically delivering IL-15 coupled or not with its soluble receptor IL-15Rα. Despite the lack of effects on myofibroblast accumulation, both IL-15 treatments prevented tubulointerstitial fibrosis (TIF) in UUO as characterized by reduced collagen and fibronectin deposition. Moreover, IL-15 treatments inhibited collagen and fibronectin secretion by transforming growth factor-ß (TGF-ß)-treated primary myofibroblast cultures, demonstrating that the antifibrotic effect of IL-15 in UUO acts, in part, through a direct inhibition of ECM synthesis by myofibroblasts. In addition, IL-15 treatments resulted in decreased expression of monocyte chemoattractant protein 1 (MCP-1) and subsequent macrophage infiltration in UUO. Taken together, our study highlights a major role of IL-15 on myofibroblasts and macrophages, two main effector cells in renal fibrosis, demonstrating that IL-15 may represent a new therapeutic option for CKD.

Adrenomedullin Ameliorates Pulmonary Fibrosis by Regulating TGF-ß-Smads Signaling and Myofibroblast Differentiation.

Pulmonary fibrosis is an irreversible, potentially fatal disease. Adrenomedullin (AM) is a multifunctional peptide whose activity is regulated by receptor activity-modifying protein 2 (RAMP2). In the present study, we used the bleomycin (BLM)-induced mouse pulmonary fibrosis model to investigate the pathophysiological significance of the AM-RAMP2 system in the lung. In heterozygous AM knockout mice (AM+/-), hydroxyproline content and Ashcroft scores reflecting the fibrosis severity were significantly higher than in wild-type mice (WT). During the acute phase after BLM administration, FACS analysis showed significant increases in eosinophil, monocyte, and neutrophil infiltration into the lungs of AM+/-. During the chronic phase, fibrosis-related molecules were upregulated in AM+/-. Notably, nearly identical changes were observed in RAMP2+/-. AM administration reduced fibrosis severity. In the lungs of BLM-administered AM+/-, the activation level of Smad3, a receptor-activated Smad, was higher than in WT. In addition, Smad7, an antagonistic Smad, was downregulated and microRNA-21, which targets Smad7, was upregulated compared to WT. Isolated AM+/- lung fibroblasts showed less proliferation and migration capacity than WT fibroblasts. Stimulation with TGF-ß increased the numbers of α-SMA-positive myofibroblasts, which were more prominent among AM+/- cells. TGF-ß-stimulated AM+/- myofibroblasts were larger and exhibited greater contractility and extracellular matrix production than WT cells. These cells were α-SMA (+), F-actin (+), and Ki-67(-) and appeared to be nonproliferating myofibroblasts (non-p-MyoFbs), which contribute to the severity of fibrosis. Our findings suggest that in addition to suppressing inflammation, the AM-RAMP2 system ameliorates pulmonary fibrosis by suppressing TGF-ß-Smad3 signaling, microRNA-21 activity and differentiation into non-p-MyoFbs.

The Scar-in-a-Jar: In Vitro Fibrosis Model for Anti-Fibrotic Drug Testing.

Excessive deposition of type I collagen follows in the wake of chronic inflammation processes in dysregulated tissue healing and causes fibrosis that can ultimately lead to organ failure. While the development of antifibrotic drugs is targeting various upstream events in collagen matrix formation (synthesis, secretion, deposition, stabilization, remodeling), the evaluation of drug effects would use as net read-out of the above effects the presence of a deposited collagen matrix by activated cells, mainly myofibroblasts. Conventional methods comprise lengthy and labor-intensive protocols for the quantification of deposited collagen, some with sensitivity and/or specificity issues. Here we describe the Scar-in-a-Jar assay, an in vitro fibrosis model for anti-fibrotic drug testing that benefits from a substantially accelerated extracellular matrix deposition employing macromolecular crowding and a collagen-producing cell type of choice (e.g., lung fibroblasts like WI-38). The system can be aided by activating compounds such as transforming growth factor-ß1, a classical inducer of the myofibroblast phenotype in fibroblasts. Direct image analysis of the well plate not only eliminates the need for matrix extraction or solubilization methods, but also allows for direct imaging and monitoring of phenotypical markers and offers the option for high-content screening applications when adapted to well formats compatible with a screening format.

Salvianolic acid B inhibits myofibroblast differentiation and extracellular matrix accumulation in nasal polyp fibroblasts via the TGF-ß1 signaling pathway.

In the process of nasal tissue remodeling, nasal fibroblasts serve an important role via myofibroblast differentiation and the production of extracellular matrix (ECM). Nasal fibroblast abnormalities can lead to conditions such as chronic rhinosinusitis. Salvianolic acid B (Sal B), a water-soluble active pharmaceutical compound extract from the root of the traditional Chinese medicine Salvia miltiorrhiza, displays antioxidative, antiproliferative and antifibrosis properties. The present study aimed to investigate the mechanism underlying the effects of Sal B on nasal polyp fibroblast (NPF) myofibroblast differentiation and ECM accumulation. Primary NPFs were obtained from nasal polyps of patients with chronic sinusitis. The proliferative and cytotoxic effects of Sal B on NPFs were evaluated by performing the Cell Counting Kit-8 assay. The Transwell assay was conducted to assess cell migration. α-smooth muscle actin (α-SMA), TGF-ß1 receptor (TßR)-I, TßR-II, Smad2/3 mRNA and protein expression levels and (p)-Smad2/3 phosphorylation levels were measured via reverse transcription-quantitative PCR and western blotting, respectively. Type III collagen and fibronectin levels were analyzed by ELISA. The results indicated that Sal B significantly downregulated TGF-ß1-induced α-SMA, fibronectin and collagen III expression levels in NPFs. Similarly, Sal B significantly decreased TGF-ß1-induced TßR-I, TßR-II, p-Smad2/3, MMP-2 and MMP-9 mRNA and protein expression levels in NPFs. Furthermore, Sal B significantly decreased TGF-ß1-induced NPF migration. Therefore, the present study indicated that Sal B inhibited myofibroblast differentiation and ECM accumulation in nasal fibroblasts, suggesting that Sal B may inhibit nasal polyp formation via certain mechanisms.

Betulinic acid attenuated bleomycin-induced pulmonary fibrosis by effectively intervening Wnt/ß-catenin signaling.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal and progressive fibrotic lung disease lacking a validated and effective therapy. Aberrant activation of the Wnt/ß-catenin signaling cascade plays the key role in the pathogenesis of IPF. Betulinic acid is a natural pentacyclic triterpenoid molecule that has excellent antitumor and antiviral activities. HYPOTHESIS: We hypothesized that BA has an anti-pulmonary fibrosis effect mediated by the suppression of the Wnt/ß-catenin pathway. Study design Pulmonary fibrosis markers were detected in vitro and in vivo to confirm the antifibrotic effect of BA. The Wnt/ß-catenin pathway-related proteins were overexpressed to determine the effect of BA on Wnt signaling. METHODS AND RESULTS: BA dose-dependently inhibited Wnt3a-induced fibroblast activation in vitro. Moreover, BA decreased Wnt3a- and LiCl-induced transcriptional activity, as assessed by the TOPFlash assay in fibroblasts, and repressed the expression of the Wnt target genes cyclin D1, axin 2, and S100A4. Further investigation indicated that BA restrained the nuclear accumulation of ß-catenin, mainly by increasing the phospho-ß-catenin ratio (S33/S37/T41 and S45), inhibited the phosphorylation of DVL2 and LRP, and decreased the levels of Wnt3a and LRP6. In agreement with the results of the in vitro assays, the in vivo experiments indicated that BA significantly decreased bleomycin-induced pulmonary fibrosis in mice and suppressed myofibroblast activation by inhibiting Wnt/ß-catenin signaling. CONCLUSION: BA may directly interfere with the Wnt/ß-catenin pathway to subsequently repress myofibroblast activation and pulmonary fibrosis.

Inhibition of proliferation-linked signaling cascades with atractylenolide I reduces myofibroblastic phenotype and renal fibrosis.

Renal fibrosis is a frequent axis contributing to the occurrence of end-stage nephropathy. Previously, it has been reported that atractylenolide Ⅰ (ATL-1), a natural compound extracted from Atractylodes macrocephala, has anti-cancer and antioxidant effects. However, the renal anti-fibrotic effects of action remain unclear. In this study, the anti-fibrotic effects of ATL-1 were examined in fibroblasts, tubular epithelial cells (TECs) triggered by TGF-ß1 in vitro, and using a unilateral ureteral obstruction (UUO) mouse model in vivo. We found that ATL-1 represses the myofibroblastic phenotype and fibrosis development in UUO kidneys by targeting the fibroblast-myofibroblast differentiation (FMD), as well as epithelial-mesenchymal transition (EMT). The anti-fibrotic effects of ATL-1 were associated with reduced cell growth in the interstitium and tubules, leading to suppression of the proliferation-linked cascades activity consisting of JAK2/STAT3, PI3K/Akt, p38 MAPK, and Wnt/ß-catenin pathways. Besides, ATL-1 treatment repressed TGF-ß1-triggered FMD and the myofibroblastic phenotype in fibroblasts by antagonizing the activation of proliferation-linked cascades. Likewise, TGF-ß1-triggered excessive activation of the proliferation-linked signaling in TECs triggered EMT. The myofibroblastic phenotype was repressed by ATL-1. The anti-fibrotic and anti-proliferative effects of ATL-1 were linked to the inactivation of Smad2/3 signaling, partially reversing FMD, as well as EMT and the repression of the myofibroblastic phenotype. Thus, the inhibition of myofibroblastic phenotype and fibrosis development in vivo and in vitro through proliferation-linked cascades of ATL-1 makes it a prospective therapeutic bio-agent to prevent renal fibrosis.

Mechanism of turnover or persistence of radiation-induced myofibroblast in vitro.

We recently made an important discovery that radiation induces myofibroblasts, which play a role in radiation-related carcinogenesis via tumor microenvironment formation. Here, we investigated the threshold dose and the mechanisms of myofibroblast induction to assess adverse radiation effects on normal cells. Single-dose of healthy human fibroblasts in vitro promotes myofibroblast induction at high doses (≥ 5 Gy). In contrast, repeated low dose of fractionated radiation is at least equivalent to high-dose single radiation regarding myofibroblast induction. ROS play a pivotal role in the process of myofibroblast induction in normal tissue injury. Antioxidants, such as epicatechin and ascorbic acid can prevent myofibroblast induction by scavenging ROS. We further investigated the role of DNA damage responses (DDR) on myofibroblast induction. Blocking the DDR using DNA-PK or AKT inhibitors enhanced cellular sensitivity to radiation and facilitated myofibroblast induction, whereas an ATM inhibitor also enhanced radiation sensitivity but abrogated ROS accumulation and myofibroblast induction. In contrast to standard culture conditions, myofibroblasts remained after low or moderate doses of radiation (below 2.5 Gy) under growth-restricted conditions. In conclusion, the recovery of damaged cells from radiation is essential for myofibroblast clearance, which restores stromal cell dormancy and prevents tumor microenvironment formation. However, residual ROS, by way of sustaining myofibroblast presence, can facilitate tumor microenvironment formation. Targeting ROS using antioxidants is effective in the mitigation of radiation-related adverse effects, such as growth retardation and myofibroblast induction, and helps protect normal tissues.

Biochanin-A ameliorates pulmonary fibrosis by suppressing the TGF-ß mediated EMT, myofibroblasts differentiation and collagen deposition in in vitro and in vivo systems.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-ß mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease. PURPOSE: The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-ß mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects. METHODS: The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-ß to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA. RESULTS: In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-ß modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-ß1/BLM-mediated increase of TGF-ß/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations. CONCLUSION: Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-ß/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.

Vitamin D modulates E-cadherin turnover by regulating TGF-ß and Wnt signalings during EMT-mediated myofibroblast differentiation in A459 cells.

Vitamin D (VitD) has an anti-fibrotic effect on fibrotic lungs. It reduces epithelial-mesenchymal transition (EMT) on tumors. We aimed to investigate target proteins of VitD for the regression of EMT-mediated myofibroblast differentiation. A group of A549 cells were treated with 5 % cigarette smoke extract (CSE) and 5 %CSE + TGF-ß (5 ng/ml) to induce EMT. The others were treated with 50 nM VitD 30 min before %5CSE and TGF-ß treatments. All cells were collected at 24, 48 and 72 h following 5 %CSE and TGF-ß administrations. The expression of p120ctn and NEDD9 proteins acted on E-cadherin turnover in addition to activations of TGF-ß and Wnt pathways were examined in these cells and fibrotic human lungs. CSE and TGF-ß induced EMT by reducing E-cadherin, p-VDR, SMAD7 and DKK1, increasing α-SMA, p120ctn, Kaiso, NEDD9 and stimulating TGF-ß and Wnt/ß-catenin signalings in A549 cells. VitD administration reversed these alterations and regressed EMT. Co-immunoprecipitation analysis revealed p-VDR interaction with ß-catenin and Kaiso in fibrotic and non-fibrotic human lungs. VitD pre-treatments reduced TGF-ß and Wnt/ß-catenin signalings by increasing p-VDR, protected from E-cadherin degradation and led to the regression of EMT in A549 cells treated with CSE and TGF-ß. Finally, VitD supplementation combined with anti-fibrotic therapeutics can be suggested for treatment of pulmonary fibrosis, which may be developed by smoking, in cases of VitD deficiency.

Pien Tze Huang accelerated wound healing by inhibition of abnormal fibroblast apoptosis in Streptozotocin induced diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic foot ulcer is one of the most serious complications of diabetes. Effective medical treatment regarding improvement of ulcer healing in patients is essential. Pien Tze Huang (PZH), a valuable Chinese traditional medicine, has been found significant efficacy on the curing of diabetic wound in clinic recently. AIM OF THE STUDY: This work was conducted to confirm the efficacy, and compare the therapeutic effect through the oral administration and local delivery route, providing a rationale for the new PZH form development; besides, the mechanisms through which PZH promoted the wound healing was also discussed. MATERIALS AND METHODS: First, the chemical composition of PZH was characterized by 1H-NMR and HPLC. The anti-apoptosis effects of PZH on high concentration glucose injured epidermal fibroblast (HFF-1) was investigated in a dose dependent way. Then, the effects of the systematical administration of PZH, and the topical used route on excisional wounds of Streptozotocin (STZ) induced diabetic mice were compared. RESULTS: The results illustrated that PZH decreased the reactive oxygen species (ROS) levels in cells, preventing cell damage/apoptosis through an ROS/Bcl-2/Bax/Caspase-3 pathway. The in vivo study proved that topical use of PZH exceeded the systematical route both in accelerating the wound closure and improving the healing quality. Meanwhile, PZH promoted wound closure through stimulating the secretion of Col-I, decreasing fibroblast apoptosis, and enhancing myo-fibroblast differentiation, in consistent with the mechanism study in vitro. CONCLUSIONS: Local used PZH improves wound healing by inhibiting the abnormal HFF-1 apoptosis and senescence. The study held a great promise for development of a topical dosage form of PZH for diabetic wound healing.

A Novel, Pan-PDE Inhibitor Exerts Anti-Fibrotic Effects in Human Lung Fibroblasts via Inhibition of TGF-ß Signaling and Activation of cAMP/PKA Signaling.

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832-a pan-PDE inhibitor, 869-a TRPA1 modulator, and 145-a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type ß1 (TGF-ß1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-ß pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research.

Biological effects of a new ultraviolet A1 prototype based on light-emitting diodes on the treatment of localized scleroderma.

Ultraviolet A1 (UVA1 ) phototherapy (spectral range 340-400 nm) is a well-established treatment option for various skin diseases such as localized scleroderma. Recent improvements of conventional UVA1 light sources (metal-halide or fluorescent lamps) have brought attention to a new light-emitting diode (LED) technology with remarkable advantages in handling and clinical routine. This study provides a preclinical histological and molecular evaluation of an LED-based UVA1 prototype with a narrower spectral range (360-400 nm) for treating localized scleroderma. Scleroderma mouse models and fibroblasts in vitro were exposed to LED-based UVA1 phototherapy or to irradiation with a commercially available metal-halide lamp emitting low-dose (20, 40 J/cm2 ), medium-dose (60 J/cm2 ) and high-dose (80, 100 J/cm2 ) UVA1 light. Both UVA1 light sources affected inflammatory genes (IL-1α and IL-6) and growth factors (TGFß-1 and TGFß-2). Increased collagen type 1 was reduced after UVA1 phototherapy. Matrix metalloproteinase-1 was more enhanced after a medium dose of LED-based UVA1 phototherapy than after conventional treatment. In vivo, dermal thickness and the amount of collagen were reduced after both treatment methods. Remarkably, myofibroblasts were more effectively reduced by a medium dose of LED-based UVA1 phototherapy. The study indicates that LED-based UVA1 phototherapy yields similar or even better results than conventional treatment. In terms of biosafety and patient comfort, LED-based UVA1 phototherapy offers clear advantages over conventional treatment because of the use of a narrower and less harmful UVA1 spectrum, less heat generation and shorter treatment times at the same irradiation intensity. Clinical studies are required to confirm these results in patients with localized scleroderma.