Tranilast Treatment Research Articles

Tranilast Treatment Prevents Chronic Radiation-Induced Colitis in Rats by Inhibiting Mast Cell Infiltration

Introduction: Mast cells are the principal cells involved in acute and chronic colitis due to radiation, known as radiation-induced colitis (RIC). In this study, we investigated whether pretreatment with tranilast, a mast cell inhibitor, could alleviate chronic RIC. Methods: A total of 23 Sprague-Dawley rats were randomly divided into three groups: control group (n = 5), radiation group (RG, n = 9), and tranilast-pretreated radiation group (TG, n = 9). The rats in the RG and the TG were irradiated in the pelvic area (1.5 cm from the anus) with a single dose of 20 Gy under general anesthesia. Tranilast (100 mg/kg) was administered intraperitoneally to the rats of the TG for 10 days, starting from the day of pelvic radiation. Ten weeks after radiation, the rats were euthanized. Rectal tissue samples were histologically evaluated for the total inflammation score (TIS) and mast cell count. The expression of MUC2, MUC5AC, and matrix metalloproteinase-9 (MMP-9) was also assessed immunohistochemically. Results: Both the TIS and specific components of TIS such as epithelial atypia, vascular sclerosis, and colitis cystica profunda (CCP) were significantly higher in the RG than in the TG (p = 0.02, 0.038, 0.025, and 0.01, respectively). Thein number of infiltrating mast cells was significantly higher in the RG than in the TG (median [range]: 20 [3−54] versus 6 [3−25], respectively; p = 0.034). Quantitatively, the number of MMP-9-positive cells was significantly higher in the RG (23.67 ± 19.00) than in the TG (10.25 ± 8.45) (mean ± standard deviation; p < 0.05). TIS and MMP-9 exhibited a strong association (correlation coefficient r = 0.56, p < 0.05). Immunohistochemically, the mucin-lake of CCP showed no staining for MUC5AC but was stained positive for MUC2. Conclusion: Tranilast pretreatment of chronic RIC showed an anti-inflammatory effect associated with the reduction of mast cell infiltration and MMP-9 expression.

Choreito, a Kampo medicine attenuates detrusor overactivity and bladder pain symptoms in rat tranilast-induced interstitial cystitis/bladder pain syndrome-like model.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory condition of the bladder. However, there are only a few medicines that are of pharmaceutical grade and reliably effective for IC/BPS symptoms. Choreito (CRT) is a pharmaceutical-grade Kampo medicine and has been widely prescribed for patients of lower urinary tract symptoms (LUTS) and BPS in Japan. In this study, we exploratory investigated the effects of CRT on the IC/BPS-like symptoms induced by tranilast. The rat IC/BPS-like model was induced by feeding administration with 0.4% tranilast. The rats were divided into the three following treatment groups: normal diet (Normal), tranilast treatment (Control), and the groups of 1% CRT (CRT) treatment for IC/BPS-like model. After 4 weeks, continuous cystmetry, locomotor, and vascular permeability was assessed. Furthermore, the cytokine levels in bladder were analyzed by the Bio-Plex suspension array system and plasma monoamine were measured. Control group exhibited 14.3% decrease of locomotor activity in the dark period, and which were 20.3% increase by 1%CRT treatment. The voiding interval was shorter in control than in other groups. 1%CRT suppressed the shortening of voiding interval. Evans blue leakage of bladder wall observed 44.8% higher in control group than in the normal group. The leakage of 1%CRT group was 33.3% less than in the control group. The cytokine level of IFNγ and VEGF were elevated in the control, and CRT treatment suppressed the elevation of IFNγ in the bladder. Plasma noradrenaline was significantly reduced by CRT treatment compared normal group. These results suggest that CRT can be an effective therapeutic agent for the treatment of IC/BPS-like symptoms.

Preparation of Micelles Encapsulating Doxorubicin and Their Anticancer Effect in Combination With Tranilast in 3D Spheroids and In Vivo.

The objective of this study was to prepare doxorubicin encapsulated in micelles (DOX-micelles) using poly(hexadecanyloxyethylene glycol-lactate phosphate), which we recently synthesized, and to evaluate the anticancer effect of DOX-micelles in vitro and in vivo. To evaluate the anticancer effect of DOX-micelles in vitro, three-dimensional spheroids composed of B16 mouse melanoma cells and fibroblasts were prepared by changing the ratio of cancer cells to fibroblasts. In addition, for efficient doxorubicin treatment of the cells present in the center of the spheroids, tranilast, an anti-fibrotic drug was added to the spheroids before treatment with DOX-micelles, then the amount of doxorubicin and cell viability of spheroids were evaluated. Moreover, to assess the effects of the combination of DOX-micelles with tranilast in vivo, relative tumor volume was investigated in a mouse model of melanoma. The mean diameter and doxorubicin content of DOX-micelles were 93.3 nm and 3.5%, respectively. When the ratio of cancer cells to fibroblasts was 20:80, spheroids with spherical and rigid shapes were obtained. In addition, the amount of doxorubicin in the spheroids was increased by tranilast treatment, and an efficient anticancer effect was also observed. The anticancer effect of the combination of tranilast and DOX-micelles was confirmed in vivo. Micelles encapsulating doxorubicin are promising for cancer therapy, and their anticancer effect is improved by tranilast pretreatment in 3D spheroids in vivo.

NLRP3 inflammasome inhibitor ameliorates ischemic stroke by reprogramming the phenotype of microglia/macrophage in a murine model of distal middle cerebral artery occlusion

Stroke is one of the leading causes of death and disability worldwide. NLRP3 inflammasome has an essential role in the neuropathology of stroke. Recent studies report that shifting the microglial M1 phenotype to the M2 phenotype protects against ischemic stroke. In the present study, the precise effects of Tranilast, a NLPR3 inflammasome inhibitor, on stroke were evaluated. We established a murine model of distal middle cerebral artery occlusion (dMCAO) and administered Tranilast to dMCAO-induced stroke mice. The NLRP3 level, caspase 1 activity, and infarct volume stroke mice were measured. The sensorimotor function, pro-inflammatory cytokine production, and M1/M2 marker expression were measured. The M1 phenotype was induced by treatment of BV2 microglia with lipopolysacharide and interferon γ, and these BV-2 cells were further treated with Tranilast. The expression of CD16 and CD206 was monitored. dMCAO increased the NLRP3 expression and enhanced caspase 1 activity. Tranilast treatment significantly decreased the infarct volume, improved sensorimotor function, and suppressed the production of inflammatory cytokines in stroke mice. Moreover, Tranilast decreased the M1 marker level while promoting the expression of M2 markers. In summary, our findings suggest that Tranilast ameliorates ischemic stroke through stimulating M2 polarization of microglia.

In vitro and vivo study of tranilast protects from acute respiratory distress syndrome and early pulmonary fibrosis induced by smoke inhalation

BackgroundTranilast (N-[3＇,4＇-dimethoxycinnamoyl]-anthranilic acid) is an analog of a tryptophan metabolite. It was identified with anti-inflammatory and antifibrotic activities, and used in the treatment of a variety of diseases, such as anti - allergy, bronchial asthma, and hypertrophic scars. As a drug with few adverse reactions, tranilast has attracted great attention, but its application is limited due to the uncertainty of dosages and mechanisms. In this study, the protection effects of different doses of tranilast on smoke inhalation mediated lung injury on rats, and on the damage of three kinds of lung cells in vitro were investigated. MethodIn vivo, Sprague-Dawley rats were randomly divided into sham group, smoke group (rats were exposed to pine sawdust smoke three times, each time for 5 min), different doses of tranilast treatment group (doses were 100 mg/kg, 200 mg/kg and 300 mg/kg, ip.) and placebo group. After 1, 3 and 7 days, pulmonary function, pathologic injury by HE staining, cytokines and oxidative stress level by kits were determined. At 7days, lung fibrosis was assessed by Masson’s trichrome staining and the level of hydroxyproline (HYP). In vitro, three kinds of lung cells from normal rats were isolated: type II alveolar epithelial cells (AT-II), pulmonary microvascular endothelial cells (PMVECs) and pulmonary fibroblasts (PFs). To investigate the potential effects of tranilast on cell proliferation, cell cycle and cytokine production of three kinds of lung cells exposed to smoke. ResultsCompared with smoke group and placebo group, tranilast treatment significantly reduced histopathological changes (such as pulmonary hemorrhage, edema and inflammatory cell infiltration, etc.), significantly reduced histopathological score (p < 0.05), increased arterial oxygen partial pressure, and decreased the levels of IL-1β, TNF-α, TGF-β1 (p < 0.05), oxidative stress and the expression of nuclear transcription factor κB (NF-κB) smoke exposed rats (p < 0.01). In particular, the effect of 200 mg/kg dose was more prominent. In vitro, smoke induced AT-II and PMVECs apoptosis, improved PFs proliferation (p < 0.01), activity of SOD and decreased the content of MDA (p < 0.01). However, tranilast seems to be turning this trend well. The inflammatory factor IL-11β, TNF-α and TGF-β1, and the expression of NF-κB were significantly lower in the tranilast treatment than in the smoke group (p < 0.01). ConclusionThis study indicates that tranilast had a protective effect on acute respiratory distress syndrome and early pulmonary fibrosis of rats in vivo. In addition, tranilast promotes proliferation of AT-II and PMVECs but inhibits PFs proliferation, down-regulates secretion of inflammatory cytokines and alleviates oxidative stress of AT-II, PMVECs and PFs after smoke stimuli in vitro.

&lt;p&gt;Tranilast Inhibits Pulmonary Fibrosis by Suppressing TGFβ/SMAD2 Pathway&lt;/p&gt;

PurposeIdiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified.MethodsWe investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin.ResultsTreatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice.ConclusionThese findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Tranilast Inhibits TGF-β1-induced Epithelial-mesenchymal Transition and Invasion/Metastasis via the Suppression of Smad4 in Human Lung Cancer Cell Lines.

Transforming growth factor β1 (TGF-β1) is an important epithelial-mesenchymal transition (EMT) activator that regulates the expression of E-cadherin and vimentin through Smad signalling. Tranilast is an anti-allergic drug that inhibits TGF-β1, and is used in the treatment of keloids and hypertrophic scars. We investigated whether tranilast inhibits TGF-β1-induced EMT and invasiveness in human non-small cell lung cancer cell lines. We examined the effects of tranilast treatment on EMT markers, TGF-β1/Smad signalling, and cell invasiveness in A549 and PC14 cells. Tumours from a mouse orthotopic lung cancer model with or without tranilast treatment were also immunohistochemically evaluated. Tranilast increased E-cadherin expression via Smad4 suppression and inhibited cell invasion in TGF-β1-stimulated cells. Tranilast treatment of the in vivo mouse model reduced the pleural dissemination of cancer cells and suppressed vimentin and Smad4 expression. Tranilast inhibited TGF-β1-induced EMT and cellular invasion/metastasis by suppressing Smad4 expression in cancer cells.

Novel Role for Tranilast in Regulating NLRP3 Ubiquitination, Vascular Inflammation, and Atherosclerosis.

BackgroundAberrant activation of the NLRP3 (nucleotide‐binding oligomerization domain, leucine‐rich repeat–containing receptor family pyrin domain‐containing 3) inflammasome is thought to play a causative role in atherosclerosis. NLRP3 is kept in an inactive ubiquitinated state to avoid unwanted NLRP3 inflammasome activation. This study aimed to test the hypothesis that pharmacologic manipulating of NLRP3 ubiquitination blunts the assembly and activation of the NLRP3 inflammasome and protects against vascular inflammation and atherosclerosis. Since genetic studies yielded mixed results about the role for this inflammasome in atherosclerosis in low‐density lipoprotein receptor– or apolipoprotein E–deficient mice, this study attempted to clarify the discrepancy with the pharmacologic approach using both models.Methods and ResultsWe provided the first evidence demonstrating that tranilast facilitates NLRP3 ubiquitination. We showed that tranilast restricted NLRP3 oligomerization and inhibited NLRP3 inflammasome assembly. Tranilast markedly suppressed NLRP3 inflammasome activation in low‐density lipoprotein receptor– and apolipoprotein E–deficient macrophages. Through reconstitution of the NLRP3 inflammasome in human embryonic kidney 293T cells, we found that tranilast directly limited NLRP3 inflammasome activation. By adopting different regimens for tranilast treatment of low‐density lipoprotein receptor– and apolipoprotein E–deficient mice, we demonstrated that tranilast blunted the initiation and progression of atherosclerosis. Mice receiving tranilast displayed a significant reduction in atherosclerotic lesion size, concomitant with a pronounced decline in macrophage content and expression of inflammatory molecules in the plaques compared with the control group. Moreover, tranilast treatment of mice substantially hindered the expression and activation of the NLRP3 inflammasome in the atherosclerotic lesions.ConclusionsTranilast potently enhances NLRP3 ubiquitination, blunts the assembly and activation of the NLRP3 inflammasome, and ameliorates vascular inflammation and atherosclerosis in both low‐density lipoprotein receptor– and apolipoprotein E–deficient mice.

Tranilast Treatment Attenuates Cerebral Ischemia-Reperfusion Injury in Rats Through the Inhibition of Inflammatory Responses Mediated by NF-κB and PPARs.

Ischemia‐reperfusion injury (IRI) occurs when blood supply returns to tissue after interruption, which is associated with life‐threatening inflammatory response. Tranilast is a widely used antiallergic agent in the treatment against bronchial asthma and keloid. To study the function of tranilast, we used IRI in rat models. The brain tissues of IRI rats with or without tranilast treatment were collected. Neuronal apoptosis in the brain was detected by terminal deoxynucleotidyl transferase nick end labeling assay, and proinflammatory cytokine levels were measured by quantitative real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay. The expression levels of nuclear factor‐kappa B (NF‐κB), inhibitor of κB (IκB) and peroxisome proliferator‐activated receptors (PPARs) were detected by Western blot. The results showed that tranilast treatment reduced neuronal apoptosis in the brain of IRI rats. Tranilast enhanced the short‐term memory and long‐term memory to novel object recognition paradigm. Tranilast treatment decreased the messenger RNA (mRNA) and protein levels of multiple proinflammatory cytokines, and affected NF‐κB and inhibitor of kappa B protein expressions. Tranilast promoted the expressions of PPAR‐α and PPAR‐γ. Our findings demonstrate that tranilast treatment could attenuate cerebral IRI by regulating the inflammatory cytokine production and PPAR expression. Tranilast is a potential drug for IRI treatment in the clinic.

Inhibitory effect of tranilast on the myofibroblast differentiation of rat mesenchymal stem cells induced by transforming growth factor‑β1 invitro.

Mesenchymal stem cell (MSC) transplantation is able to attenuate organ fibrosis; however, increasing evidence has indicated that MSCs may be an important cell source of myofibroblasts, which are vital pathogenic cells in fibrotic diseases. The results of the present study revealed that co‑culturing with exogenous transforming growth factor (TGF)‑β1 can induce the transdifferentiation of cultured rat MSCs into myofibroblasts invitro. Treatment of the MSCs with tranilast [N‑(3',4'‑dimethoxycinnamoyl)‑anthranilic acid] attenuated this fibrotic process. Immunocytochemical staining, western blot analysis, reverse transcription‑quantitative polymerase chain reaction analysis and cell viability assays were performed in order to evaluate the molecular mechanisms underlying the effects of tranilast on TGF‑β1‑mediated MSC‑to‑myofibroblast activation. The results demonstrated that TGF‑β1 upregulated the expression of α‑smooth muscle actin (α‑SMA) and collagen type I, and increased the phosphorylation of mothers against decapentaplegic homolog3 (Smad3) and extracellular signal‑regulated kinase1/2 (ERK1/2) in the rat MSCs; by contrast, tranilast pretreatment downregulated their expression. Furthermore, the proliferation of MSCs induced by TGF‑β1 was decreased by pretreatment with tranilast. In conclusion, the results of the present study demonstrated that tranilast treatment markedly suppressed the TGF‑β1‑induced differentiation of cultured rat MSCs into myofibroblasts, potentially by inhibiting the Smad3 and ERK1/2 signaling pathways. Therefore, this may be a potential antifibrotic therapeutic strategy, serving as an adjuvant treatment following transplantation of MSCs.

Tranilast inhibits TGF-β-induced collagen gel contraction mediated by human corneal fibroblasts.

To determine if tranilast affects human corneal fibroblast (HCFs) contraction. HCFs cultured in a three-dimensional type I collagen gel were treated with or without transforming growth factor beta (TGF-β) or tranilast. Gel diameter was measured as an indicator for collagen contraction. Immunoblot was performed to evaluate myosin light chain (MLC) and paxillin phosphorylation. Confocal microscopy was employed to examine the focal adhesions and actin stress fiber formation. Immunoblot analysis and gelatin zymography were performed to detect tissue inhibitors of metalloproteinases and matrix metalloproteinases (MMPs) in supernatant. The inhibitory effect of tranilast on HCFs-mediated collagen gel contraction induced by TGF-β was dose-dependent. The significant effect of tranilast was started from 100 µmol/L and maximized at 300 µmol/L. The peak effect of 300 µmol/L tranilast also relied on the duration of treatment, which showed statistical significance from day 2. TGF-β-induced paxillin and MLC phosphorylation, stress fiber formation, focal adhesions, and MMP-1, MMP-2, and MMP-3 secretion in HCFs were also inhibited by tranilast. Tranilast suppresses the HCFs-cultured collagen gel contraction induced by TGF-β. It attenuates actin stress fibers formation, focal adhesions, and the secretion of MMPs, with these actions likely contributing to the inhibitory effect on HCF contractility. By attenuating the contractility of corneal fibroblasts, tranilast treatment may inhibit corneal scarring.

Tranilast prevents renal interstitial fibrosis by blocking mast cell infiltration in a rat model of diabetic kidney disease.

Renal interstitial fibrosis is a final pathway that is observed in various types of kidney diseases, including diabetic kidney disease (DKD). The present study investigated the effect of tranilast on renal interstitial fibrosis and the association between its role and mast cell infiltration in a rat model of DKD. A total of 30 healthy 6‑week‑old male Sprague‑Dawley rats were randomly divided into the following four groups: Normal control group; DKD model group; low‑dose tranilast group (200mg/kg/day); and high‑dose tranilast group (400mg/kg/day). The morphological alterations of tubulointerstitial fibrosis were evaluated by Masson's trichrome staining, while mast cell infiltration into the renal tubular interstitium was measured by toluidine blue staining and complement C3a receptor 1 (C3aR) immunohistochemical staining (IHC). The expression of fibronectin (FN), collagen I (Col‑I), stem cell factor (SCF) and proto‑oncogene c‑kit (c‑kit) was detected by IHC, western blotting and reverse transcription‑quantitative‑polymerase chain reaction. The results demonstrated that tubulointerstitial fibrosis and mast cell infiltration were observed in DKD model rats, and this was improved dose‑dependently in the tranilast treatment groups. The expression of FN, Col‑I, SCF and c‑kit mRNA and protein was upregulated in the tubulointerstitium of DKD model rats compared with the normal control rats, and tranilast inhibited the upregulated expression of these markers. Furthermore, the degree of SCF and c‑kit expression demonstrated a significant positive correlation with C3aR‑positive mast cells and the markers of renal interstitial fibrosis. The results of the present study indicate that mast cell infiltration may promote renal interstitial fibrosis via the SCF/c‑kit signaling pathway. Tranilast may prevent renal interstitial fibrosis through inhibition of mast cell infiltration mediated through the SCF/c-kit signaling pathway.

Importance of human peritoneal mesothelial cells in the progression, fibrosis, and control of gastric cancer: inhibition of growth and fibrosis by tranilast

BackgroundScirrhous gastric cancer is an intractable disease with a high incidence of peritoneal dissemination and obstructive symptoms (e.g., ileus, jaundice, and hydronephrosis) arising from accompanying marked fibrosis. Microenvironmental interactions between cancer cells and cancer-associated fibroblasts are the suggested cause of the disease. We elucidated the mechanisms of tumor growth and fibrosis using human peritoneal mesothelial cells (HPMCs) and investigated the effects of tranilast treatment on cells and a xenograft mouse model of fibrosis.MethodsHPMCs were isolated from surgically excised omentum and their interaction with MKN-45 gastric cancer cells was investigated using co-culture. Furthermore, a fibrosis tumor model was developed based on subcutaneous transplantation of co-cultured cells into the dorsal side of nude mice to form large fibrotic tumors. Mice were subsequently treated with or without tranilast.ResultsThe morphology of HPMCs treated with transforming growth factor (TGF)-β1 changed from cobblestone to spindle-type. Moreover, E-cadherin was weakly expressed whereas high levels of α-smooth muscle actin expression were observed. TGF-β-mediated epithelial–mesenchymal transition-like changes in HPMCs were inhibited in a dose-dependent manner following tranilast treatment through inhibition of Smad2 phosphorylation. In the mouse model, tumor size decreased significantly and fibrosis was inhibited in the tranilast treatment group compared with that in the control group.ConclusionsTranilast acts on the TGF-β/Smad pathway to inhibit interactions between cancer cells and cancer-associated fibroblasts, thereby inhibiting tumor growth and fibrosis. This study supports the hypothesis that tranilast represents a novel strategy to prevent fibrous tumor establishment represented by peritoneal dissemination.

Protective effects of tranilast on oxazolone-induced rat colitis through a mast cell-dependent pathway

BackgroundMast cells in the gut play an important role in the innate and adaptive immune responses that are relevant to human inflammatory bowel disease. However, the contribution of mast cells to the development of inflammatory bowel disease is not well understood. This study aimed to determine the role of mast cells in oxazolone-induced colitis and to explore whether the mast cell membrane stabiliser tranilast could ameliorate colonic inflammation. MethodsWild-type rats and mast cell-deficient rats were sensitised and challenged with oxazolone, then treated with tranilast after challenge. Controls were treated with saline. ResultsMast cell-deficient rats presented a weak response to oxazolone, while wild-type rats showed severe ulcerative colitis after stimulation with oxazolone. The mast cell-deficient rats model had a significantly lower disease activity index score than wild-type rats model (1.8±1.64 vs 8.3±0.58 respectively; P<0.01). Tranilast could reduce the secretion of cytokines, immunoglobulins and myeloperoxidase activity in tranilast treatment groups compared with the model group. The number of mast cells in the wild-type model was higher than in the other groups. There was no significant change in mast cell-deficient rats. ConclusionMast cells play an important role in oxazolone-induced colitis. The mast cell membrane stabiliser tranilast can ameliorate oxazolone-induced colitis via a mast cell-dependent pathway.

Tranilast increases vasodilator response to acetylcholine in rat mesenteric resistance arteries through increased EDHF participation.

Background and PurposeTranilast, in addition to its capacity to inhibit mast cell degranulation, has other biological effects, including inhibition of reactive oxygen species, cytokines, leukotrienes and prostaglandin release. In the current study, we analyzed whether tranilast could alter endothelial function in rat mesenteric resistance arteries (MRA).Experimental ApproachAcetylcholine-induced relaxation was analyzed in MRA (untreated and 1-hour tranilast treatment) from 6 month-old Wistar rats. To assess the possible participation of endothelial nitric oxide or prostanoids, acetylcholine-induced relaxation was analyzed in the presence of L-NAME or indomethacin. The participation of endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced response was analyzed by preincubation with TRAM-34 plus apamin or by precontraction with a high K+ solution. Nitric oxide (NO) and superoxide anion levels were measured, as well as vasomotor responses to NO donor DEA-NO and to large conductance calcium-activated potassium channel opener NS1619.Key ResultsAcetylcholine-induced relaxation was greater in tranilast-incubated MRA. Acetylcholine-induced vasodilation was decreased by L-NAME in a similar manner in both experimental groups. Indomethacin did not modify vasodilation. Preincubation with a high K+ solution or TRAM-34 plus apamin reduced the vasodilation to ACh more markedly in tranilast-incubated segments. NO and superoxide anion production, and vasodilator responses to DEA-NO or NS1619 remained unmodified in the presence of tranilast.Conclusions and ImplicationsTranilast increased the endothelium-dependent relaxation to acetylcholine in rat MRA. This effect is independent of the nitric oxide and cyclooxygenase pathways but involves EDHF, and is mediated by an increased role of small conductance calcium-activated K+ channels.

Tranilast Alleviates Endothelial Dysfunctions and Insulin Resistance via Preserving Glutathione Peroxidase 1 in Rats Fed a High-Fat Emulsion

We investigated the effects of treatment with tranilast on vascular and metabolic dysfunction induced by a high-fat emulsion intragastric administration. Wistar rats were randomized to receive water or high-fat emulsion with or without tranilast treatment (400 mg/kg per day) for 4 weeks. Insulin sensitivity was determined with a hyperinsulinemic-euglycemic clamp experiment and short insulin tolerance test. Vascular reactivity was evaluated using aortic rings in organ chambers. Glutathione peroxidase 1 (GPX1) expressions, eNOS phosphorylation and activity, MCP-1, H2O2 formation, and NO production were determined in vascular or soleus tissues. Tranilast treatment was found to prevent alterations in vascular reactivity and insulin sensitivity and to prevent increases in plasma glucose and insulin noted in the high-fat emulsion-treated rats. These were associated with increased antioxidant enzyme GPX1 expression, eNOS phosphorylation and activity, and NO production, but reductions in H2O2 accumulation. Moreover, tranilast preserved GPX1 expression in palmitic acid (PA)-treated endothelial cells with a consequent decreased ROS formation and increased eNOS phosphorylation and NO production. Therefore, oxidative stress induced by a relatively short-term high-fat diet could cause the early development of vascular and metabolic abnormalities in rats, and tranilast has a beneficial effect in vascular dysfunctions and insulin resistance via preserving GPX1 and alleviating oxidative stress.

Role and mechanism of tranilast preventing the progression of tubulointerstilial fibrosis in diabetic kidney diseases

To determine the role and mechanism of tranilast preventing the progression of tubulointerstilial fibrosis in diabetic kidney disease (DKD). Sprague-Dawley rats were randomly divided into a control group (n=6), DKD model group (n=8), low dose tranilast group [200 mg/(kg.d), n=8], and high dose tranilast group [400 mg/(kg.d), n=8]. Tranilast was administered daily after the model was built. Rats were sacrificed at day 56, 24 hour urine was collected to measure 24-hour urine albumin excretion, and blood was collected to determine the renal function and serum albumin. Then the kidneys were harvested and subjected to studies. The expression of C3aR, E-cadherin, α-SMA, fibronectin(FN), collagen I (Col I), stem cell factor (SCF) and c-kit were detected by immunohistochemical staining respectively. The expression of E-cadherin, α-SMA, FN, Col I, SCF and c-kit protein was analyzed by Western blot, and the expression of FN, Col I, SCF and c-kit mRNA was examined by RT-PCR. Tranilast can inhibit the infiltration of mast cells in the kidneys of DKD rats. The expression of α-SMA in the kidneys of DKD rats inereased significantly (P<0.05), while the expression of E-cadherin decreased (P<0.05). Tranilast increased the expression of E-cadherin and decreased the expression of α-SMA in the prophase of DKD dose dependently. The expressions of FN and Col I were increased in the tubulointerstitial fields in DKD model rats (P<0.05). After the tranilast treatment, these changes were relieved to a certein degree (P<0.05). The expression of SCF and c-kit in the tubular and interstitial tissue was slight. The increased expressions of SCF and c-kit protein and mRNA in DKD model rats were downregulated by tranilat (P<0.05). The expressions of SCF and c-kit were positively correlated with the infiltration degree of mast cells and the expressions of FN, Col I. Mast cells participate in and aggravate the renal tubulointerstitial fibrosis in DKD rats. Tranilast can reverse the EMT of renal tubular cells and inhibit the tubulointersitial fibrosis of DKD by blocking the infiltration of mast cells induced by SCF/c-kit pathway.

Synergistic Effects of Tamoxifen and Tranilast on VEGF and MMP-9 Regulation in Cultured Human Breast Cancer Cells

Vascular endothelial growth factor and matrix metalloproteinases are two important factors for angiogenesis associated with breast cancer growth and progression. The present study was aimed to examine the effects of tamoxifen and tranilast drugs singly or in combination on proliferation of breast cancer cells and also to evaluate VEGF and MMP-9 expression and VEGF secretion levels. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were treated with tamoxifen and/or tranilast alone or in combination and percentage cell survival and proliferative activity were evaluated using LDH leakage and MTT assays. mRNA expression and protein levels were examined by real-time RT-PCR and ELISA assay, respectively. LDH and MTT assays showed that the combined treatment of tamoxifen and tranilast resulted in a significant decrease in cell viability and cell proliferation compared with tamoxifen or tranilast treatment alone, with significant decrease in VEGF mRNA and protein levels. We also found that tamoxifen as a single agent rarely increased MMP-9 expression. A decrease in MMP-9 expression was seen after treatment with tranilast alone and in the combined treatment MMP-9 mRNA level was decreased. This combination treatment can able to inhibit growth, proliferation and angiogenesis of breast cancer cells.

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

ObjectivesWe analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.MethodsElectrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O2 .-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.ResultsEFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O2 - release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.ConclusionWhile both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.

Abstract 5510: Tranilast inhibits growth of skin tumor cells in a xenograft mouse model of tuberous sclerosis complex.

Abstract Pharmacological approaches for treating tumors in patients with tuberous sclerosis complex (TSC) are inadequate. Although mTORC1 inhibitors such as rapamycin represent a major new avenue for treatment, we and others have shown that rapamycin improves but does not eradicate TSC skin tumors. We tested the effects of tranilast, an antiallergic and antifibrotic drug with antiproliferative effects, on fibroblasts grown from angiofibromas, forehead plaques, periungual fibromas, and normal-appearing skin from 5 patients with TSC. Tumor cells showed loss of TSC2 expression and hyperactivation of the mTORC1 signaling pathway. Tranilast (5, 10, 20, 40 μM) inhibited the viability of TSC skin tumor cells from five patients in an MTT assay. These effects on cell viability appeared to be independent of mTORC1 activity, since tranilast did not inhibit phosphorylation of ribosomal protein S6. Four paired samples of RNA from tumor and normal cells, treated with or without tranilast, were studied using gene expression arrays, revealing that tranilast significantly altered the expression of at least 36 genes, of which 12 are involved in transcription. In a xenograft model in which dermal-epidermal constructs comprising normal human keratinocytes with either TSC angiofibroma cells or TSC normal fibroblasts from a TSC patient were grafted to nude mice (6-8 mice per group), tranilast treatment (300 mg/kg/day) reduced the number of skin tumor cells compared to vehicle control group (P&amp;lt; 0.05). Tranilast treatment did not block angiogenesis or lymphangiogenesis in the xenograft model. These results indicate that tranilast has antiproliferative effects independent of mTORC1 and suggest that tranilast may be useful as an adjunctive agent for the treatment of TSC skin tumors. Citation Format: Shaowei Li, Ji-an Wang, Rajesh L. Thangapazham, Peter Klover, Joel Moss, Thomas N. Darling. Tranilast inhibits growth of skin tumor cells in a xenograft mouse model of tuberous sclerosis complex. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2013-5510