Epigallocatechin gallate attenuates fibroblast proliferation and excessive collagen production by effectively intervening TGF-ß1 signalling.

Pulmonary fibrosis (PF) poses a huge burden to the patients and society due to lack of an effective treatment drug. Activation of fibrocyte, fibroblast and myofibroblasts are important steps in the development of PF. Targeting this common pathway with natural chemicals may lead to the development of new drug regimens for PF treatment. In this study, PF was induced in male Wistar rats by intratracheal administration of Bleomycin (BLM). Epigallocatechin gallate (EGCG) was administered to one of the groups of rats to test its efficacy against the development of PF. Bleomycin-induction resulted in significant elevation of matrix metalloproteinase (MMP)-2 and -9 expression, increased RNA and protein expression of transforming growth factor (TGF)-ß1, Smads and alpha-smooth muscle actin (α-SMA). EGCG treatment normalized the BLM induced aberrations in these rats. The protective role of EGCG was also validated in vitro using the WI-38 fibroblast cell line. TGF-ß1 incubated cells exhibited increased fibroblast proliferation and hydroxyproline levels with a concomitant decrease in the expression of MMPs 2 and 9. An increase in protein expression levels of p-Smad, α-SMA and type I collagen (COL1A) was also exhibited by fibroblasts upon TGF-ß1 incubation. Simultaneous treatment of EGCG to WI-38 cells significantly decreased these protein expressions alongside normalizing the MMPs expression. The study revealed that EGCG inhibited fibroblast activation and collagen accumulation by inhibiting TGF-ß1 signalling and thus can be considered as an effective drug against PF.

Daidzein exhibits anti-fibrotic effect by reducing the expressions of Proteinase activated receptor 2 and TGFß1/smad mediated inflammation and apoptosis in Bleomycin-induced experimental pulmonary fibrosis.

Pulmonary fibrosis (PF) is a progressive lethal disorder. In this study, the effect of daidzein, a soyisoflavone against Bleomycin (BLM) induced PF in rats was elucidated. A single intratracheal instillation of BLM (3 U/kg.bw) was administered in rats to induce PF. Daidzein (0.2 mg/kg) was administered subcutaneously, twice a week for a period of 28 days. Daidzein restored the histological alteration and aberrant collagen deposition, suppressed the mast cells, and reduced the expressions of Cyclooxygenase 2 (COX2) and Nuclear factor kappa B (Nf-kB) in lung tissue of BLM-induced rats. Treatment with daidzein reduced the expression of Matrix metalloproteinase 2 (MMP-2) and increased the expression of Tissue inhibitor of matrixmetalloproteinases 1 (TIMP 1). Recently, Proteinase activated receptor 2 (PAR2) has been reported to play a major role in the progression of PF. Confocal microscopic and immunoblot analysis revealed that BLM injured rat lungs exhibited increased expression of PAR2 that was reduced upon treatment with daidzein. During BLM induction, Transforming growth factor beta (TGFß1) was found to be up-regulated along with p-smad2/3, a mediator of TGFß signaling. Further, daidzein regulated the apoptosis by modulating the expressions of Bcl-2, Bax and caspase 3. This study provides evidence on the anti-fibrotic role of daidzein in BLM-induced experimental fibrosis.

Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2.

Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF.

TC-PTP dephosphorylates the guanine nucleotide exchange factor C3G (RapGEF1) and negatively regulates differentiation of human neuroblastoma cells.

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.

Epigallocatechin-3-gallate exhibits anti-fibrotic effect by attenuating bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in rat model pulmonary fibrosis.

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix components in the alveolar space, which hampers normal respiration process. Pathophysiological enzymes, glycoprotein moieties and matrix degrading lysosomal hydrolases are key markers and play a crucial role in the progression of fibrosis. Bleomycin is an anti-neoplastic drug, used for the treatment of various types of cancers and induces pulmonary fibrosis due its deleterious side effect. Tea catechin epigallocatechin-3-gallate (EGCG) is known for its wide array of beneficial effects. The present study was intended to evaluate the beneficial efficacy of EGCG against bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in the lungs of Wistar rats. Intratracheal instillation of bleomycin (6.5 U/kg body weight) to rats increased the activities of pathophysiological enzymes such as aspartate transaminase, alanine transaminase, lactate dehydrogenase and alkaline phosphatase, which were attenuated upon EGCG treatment. The increased level of hydroxyproline and histopathological parameters in bleomycin-induced rats were decreased upon EGCG treatment. Bleomycin-induced increase in the level of glycoconjugates was restored closer to normal levels on EGCG treatment. Furthermore, the increased activities of matrix degrading lysosomal enzymes in bleomycin-induced rats were reduced upon EGCG supplementation. Treatment with EGCG also attenuated bleomycin-induced ultrastructural changes as observed from transmission electron microscopy studies. The results of the present study put-forward EGCG as a potential anti-fibrotic agent due to its attenuating effect on potential fibrotic markers.

S-allylcysteine attenuates renal injury by altering the expressions of iNOS and matrix metallo proteinase-2 during cyclosporine-induced nephrotoxicity in Wistar rats.

Cyclosporine A (CsA) is the first choice immunosuppressant used for the prevention of allograft rejection in solid organ transplantation and immune-mediated diseases. Reactive oxygen species-induced oxidative stress and lipid peroxidation are implicated in the pathophysiology of CsA-induced renal injury. In this work, we have studied the effect of a garlic-derived compound, S-allylcysteine (SAC) on CsA-induced nephrotoxicity. CsA-induced nephrotoxicity was assessed in terms of increased activities of serum marker enzymes and levels of kidney markers. CsA administration induced significant elevation in lipid peroxidation along with abnormal levels of enzymic and non-enzymic antioxidants in the kidneys of the rats. SAC administration improved renal function by bringing about a significant decrease in peroxidative levels and increase in antioxidant status. Elevated expressions of inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-kappaB) due to CsA administration were reduced by SAC treatment. An increase in the expression of matrix metalloproteinase-2 (MMP-2) was evident in CsA-induced groups of rats, which was moderately reduced in SAC treated rats. An increase in the levels of serum constituent's urea, uric acid and creatinine was observed in the CsA-induced rats, which was reduced upon treatment with SAC. These results indicate that SAC has a protective action against CsA-induced nephrotoxicity which is also supported by histopathological studies. A comparative study of the antioxidant vitamin C and SAC is more valuable to assess the efficacy of the drug that can be used for the treatment of nephrotoxicity.

Diallyl sulfide enhances antioxidants and inhibits inflammation through the activation of Nrf2 against gentamicin-induced nephrotoxicity in Wistar rats.

The protective role of diallyl sulfide (DAS) in attenuating gentamicin-induced nephrotoxicity has been reported earlier. However, the mechanism of induction of antioxidants by DAS in nephrotoxicity remains elusive. This study is aimed to elucidate the role of a transcription factor, Nuclear factor E2-related factor 2 (Nrf2) in inducing antioxidants and phase II enzymes during gentamicin toxicity in Wistar rats. DAS was administered intraperitoneally at a dosage of 150 mg/kg body weight once daily for 6 days. Gentamicin was administered intraperitoneally at a dosage of 100 mg/kg body weight, once daily for 6 days. Gentamicin-induced rats showed a significant increase in the levels of kidney markers and the activities of urinary marker enzymes, which was reversed upon treatment with DAS. A significant increase in kidney myeloperoxidase (MPO) and lipid peroxidation (LPO) levels was observed in gentamicin-induced rats, which was reduced upon treatment with DAS. Gentamicin-induced rats also showed a significant decrease in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and quinone reductase (QR) in rat kidney, which was increased upon treatment with DAS. Immunohistochemical studies in gentamicin-induced rats demonstrated a marked increase in the immunoreactivity of inducible nitric oxide synthase (iNOS), nuclear transcription factor (NF-kappaB) and tumor necrosis factor alpha (TNF-alpha) that were reduced after treatment with DAS. Further, the involvement of Nrf2 in antioxidant induction was analyzed by Western blot and immunofluorescence. To conclude, DAS enhances antioxidants and suppresses inflammatory cytokines through the activation of Nrf2, thereby protecting the cell against oxidative stress induced by gentamicin.

Epigallocatechin-3-gallate augments antioxidant activities and inhibits inflammation during bleomycin-induced experimental pulmonary fibrosis through Nrf2-Keap1 signaling.

The mechanism involved in the enhancement of antioxidant activities and resolved inflammation after epigallocatechin-3-gallate (EGCG) treatment during bleomycin-induced pulmonary fibrosis is investigated in this study. The levels of reactive-oxygen species (ROS), lipid peroxidation (LPO), hydroxyproline and the activity of myeloperoxidase (MPO) were increased due to bleomycin challenge and were brought back to near normal status on EGCG supplementation. The decreased antioxidant status due to bleomycin challenge was also restored upon EGCG treatment. Bleomycin-induced rats showed increased cell counts as compared to control and EGCG-treated rats. Histopathological analysis showed increased inflammation and alveolar damage, while picrosirius red staining showed an increased collagen deposition in bleomycin-challenged rats that were decreased upon EGCG treatment. Immunohistochemical, immunofluorescent and immunoblot studies revealed that EGCG supplementation decreased the levels of nuclear factor-kappaB (NF-kappaB), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), which were increased upon bleomycin induction. The declined activities of Phase II enzymes such as glutathione-S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in bleomycin-injured rats were restored upon EGCG treatment. Confocal microscopy, immunoblot and RT-PCR studies confirm that EGCG is a potent inducer of NF-E2-related factor 2 (Nrf2). Expression of Kelch like ECH-associated protein (Keap)-1, a vital factor in Nrf2 signaling cascade was analyzed by immunoblotting. However, there was no significant change in the expression of Keap1 in control and experimental groups. This study demonstrates the involvement of Nrf2-Keap1 signaling through which EGCG enhances antioxidant activities and Phase II enzymes with subsequent restraint inflammation during bleomycin-induced pulmonary fibrosis.

Enhancement of antioxidant defense system by epigallocatechin-3-gallate during bleomycin induced experimental pulmonary fibrosis.

Oxidative stress resulting from an imbalance between radical-generating and radical scavenging systems plays an important role in the pathogenesis of pulmonary fibrosis. Epigallocatechin-3-gallate (EGCG), a polyphenol and a major component of green tea, possess a potent antioxidant property. This study was designed to evaluate the potential antioxidative activity of EGCG in the plasma and lungs during bleomycin induced experimental pulmonary fibrosis. Intratracheal administration of bleomycin (6.5 U/kg body weight) to rats resulted in significant reduction of body weight, enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase) and non-enzymic antioxidants (reduced glutathione, vitamin C, vitamin E and vitamin A). Elevations in lung W/D (wet weight/dry weight) ratio, hydroxyproline content was observed with a synchronized increase in lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides). Intraperitoneal administration of EGCG at a dose of 20 mg/kg body weight significantly improved the body weight, enzymic and non enzymic antioxidants and considerably decreased the W/D ratio, hydroxyproline and lipid peroxidation marker levels. Histological observations also correlated with the biochemical parameters. Thus, this study confirms the beneficial use of EGCG in alleviating the oxidative stress induced during pulmonary fibrosis.

Chromium (VI)-induced oxidative stress and apoptosis is reduced by garlic and its derivative S-allylcysteine through the activation of Nrf2 in the hepatocytes of Wistar rats.

Chromium (VI) compounds are genotoxic and carcinogenic in a variety of experimental systems. Garlic and its derivatives possess antioxidant properties to scavenge the toxic radicals. The mechanism by which garlic induces the antioxidant and phase II enzymes during oxidative stress-induced apoptosis is not known. This study aims to evaluate the protective role of aqueous garlic extract (AGE; 200 mg kg(-1) b.w.) and S-allylcysteine (SAC; 100 mg kg(-1) b.w.) on potassium dichromate-induced apoptosis and oxidative stress in the hepatocytes of Wistar rats. Activities of liver marker enzymes such as aspartate transaminase, alanine transaminase and lactate dehydrogenase were found to be increased in the serum of chromium-induced group, whereas administration of garlic extract and SAC restored the enzymes to near normal status. The activities of enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase), non-enzymic antioxidants (vitamin C and vitamin E) and the levels of reduced glutathione were found to be decreased, while an increase in lipid peroxidation (LPO) and reactive oxygen species were observed in the liver tissues of chromium-induced group. Administration of AGE and SAC reversed the status of these parameters substantially. Histological and transmission electron microscopic studies support our findings. Confocal microscopic analysis using annexin-V showed the involvement of apoptosis. Further, the expression of a novel transcription factor, nuclear factor-E2 related factor 2 (Nrf2) was investigated using Immunofluorescence and Western blotting. The results show the promising role of Nrf2-mediated antioxidant defense of AGE and SAC against chromium toxicity.

Diallyl sulfide attenuates bleomycin-induced pulmonary fibrosis: critical role of iNOS, NF-kappaB, TNF-alpha and IL-1beta.

Diallylsulfide (DAS), an antioxidant and anti-inflammatory agent was evaluated for its ability to repress lung fibrosis induced by bleomycin in Wistar rats. A single intra tracheal administration of bleomycin (6.5 U/kg BW) was administered to pulmonary fibrosis group, while DAS (120 mg/kg BW) was administered intraperitoneally throughout the experimental period. Fibrotic changes in the lungs were estimated by measuring lung hydroxyproline content. Bleomycin administration significantly (P<0.05) reduced the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the lung tissues. Bleomycin caused a significant decrease in the level of reduced glutathione (GSH), which was accompanied with significant increase in lipid peroxidation (LPO) level, and myeloperoxidase (MPO) activity, in the lung tissues. An increase in the level of cell counts in bronchoalveolar lavage fluid (BALF) was observed in bleomycin induced group. DAS administration altered the levels of enzymic antioxidants, TBARS, MPO and GSH towards normal values. Histopathological analysis and picrosirius red staining showed an increased collagen deposition in rats receiving bleomycin alone that was decreased upon DAS treatment. Immunohistochemical studies revealed that DAS reduced the bleomycin-induced activation of inducible nitric oxide synthase (iNOS) and nuclear factor kappa-B (NF-kappaB) and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), in the lung tissues. The present study provides evidence that DAS might serve as a novel target for the therapeutic treatment of lung fibrosis.

Diallyl sulfide induces apoptosis in Colo 320 DM human colon cancer cells: involvement of caspase-3, NF-kappaB, and ERK-2.

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Diallyl sulfide (DAS), an organosulfur component of garlic has been known for its chemopreventive activities against various cancers and also in recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. Thus the present study was focused to elucidate the anticancerous effect and the mode of action of DAS against Colo 320 DM colon cancer cells. DAS induced apoptosis in Colo 320 DM cells was revealed by flow cytometer analysis and phosphatidyl serine exposure. DAS also promoted cell cycle arrest substantially at G2/M phase in Colo 320 DM cells. The production of reactive oxygen intermediates, which were examined by 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA), increased with time, after treatment with DAS. The activities of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were decreased upon DAS treatment, which shows the antiproliferative and the cytotoxic effects, respectively. The expression of NF-kappaB was upregulated in DAS treated cells, compared to normal cells. Further, DAS promoted the expression of caspase-3 and suppression of Extracellular Regulatory Kinase-2 (ERK-2) activity in Colo 320 DM cells that was determined by Western blot analysis. In conclusion, DAS increased the production of ROS, caused cell cycle arrest, decreased cell proliferation and induced apoptosis in Colo 320 DM cells. Thus, this study put forward DAS as a drug that can possibly be used to treat cancers.