Morin ameliorates chemically induced liver fibrosis in vivo and inhibits stellate cell proliferation in vitro by suppressing Wnt/β-catenin signaling

The anti-fibrotic effect of morin was examined in LX-2 cells (culture-activated human hepatic stellate cells) and in diethylnitrosamine induced rat model of liver fibrosis. The in vitro study was designed to determine whether morin affects the survival of cultured LX-2 cells, while the in vivo study was designed to evaluate the antioxidant and anti-fibrotic efficacy of morin on diethylnitrosamine induced liver fibrosis in male albino Wistar rat. The activities of liver function enzymes in serum, liver lipid peroxide levels, activities of serum antioxidant enzymes and liver architecture were monitored to cast light on the antioxidant and hepatoprotective nature of morin. To establish the anti-fibrotic effects of morin, the levels of key Wnt signaling molecules which are strongly associated with the signal transduction pathway of HSC activation were measured. Overall, from the in vitro results, it was observed that morin at 50 μM concentration inhibited the proliferation of cultured LX-2 cells, inhibited Wnt signaling and induced G1 cell cycle arrest. The in vivo results further confirmed that morin by downregulating the expressions of GSK-3β, β-catenin and cyclin D1 ameliorated DEN-induced liver fibrosis. Hence morin could be employed as a promising chemopreventive natural supplement for liver fibrosis.     

Arsenic trioxide induces cardiac fibroblast apoptosis in vitro and in vivo by up-regulating TGF-β1 expression

Arsenic trioxide (As₂O₃; ATO) is clinically effective in treating acute promyelocytic leukemia (APL); however, it frequently causes cardiotoxic effects. This study was designed to investigate whether ATO could induce apoptosis of cardiac fibroblasts (CFs) that play very important roles in maintaining the structure integrity and function of the heart. Cardiac fibroblasts from guinea pigs administered with ATO (1 mg/kg bw) were used to test the pro-apoptotic role of ATO in vivo. The current study demonstrated that ATO induced morphological characteristics of apoptosis and Caspase-3 activation in CFs of guinea pigs along with a significant up-regulation in TGF-β1 protein expression, Bax/Bcl-2 ratio and ERK1/2 phosphorylation. In vitro MTT assay showed that ATO remarkably reduced the viability of cultured cardiac fibroblasts (NRCFs) from neonatal rat in a concentration- and time-dependent manner. Consistent with the notions in vivo, ATO significantly induced the apoptosis in NRCFs, dramatically up-regulated TGF-β1 protein level and Bax/Bcl-2 ratio in a time-dependent fashion and activated Caspase-3 and ERK1/2. Finally, pretreatment with LY364947, an inhibitor of TGF-β signaling could apparently reverse these changes. We therefore conclude that TGF-β is functionally linked to ERK1/2 and that TGF-β signaling is responsible for ATO-induced CFs apoptosis, which provides a novel mechanism of ATO related cardiac toxicology.     

Expression of TNF-α and IL-6 in HMC-1 cells treated with bisphenol A is attenuated by plant-originating glycoprotein (75 kDa) by blocking p38 MAPK

Bisphenol A (BPA) is known as an estrogen-mimic environmental hormone which has the ability to indirectly stimulate the production of allergic inflammation-related cytokines. Cudrania tricuspidata Bureau (CTB) has been used in Korean folk medicine for a long time. In order to determine the inhibitory effect of a glycoprotein (CTB glycoprotein, 75 kDa) isolated from CTB fruits on the activities of allergic inflammation-related cytokines (TNF-α and IL-6) caused by BPA, we evaluated the activities of protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor (NF)-κB, and inflammation-related cytokine (TNF-α and IL-6) in the BPA-induced HMC-1 cells using immunoblot analysis and RT-PCR. The results obtained from this study revealed that CTB glycoprotein (100 μg/ml) inhibits the translocation of PKC from cytosol to the membrane, the phosphorylation of p38 MAPK, the activation of NF-κB, and the expression levels of TNF-α and IL-6. Taken together, the results in this study suggest that CTB glycoprotein inhibits the expression of allergic inflammation-related cytokines (TNF-α and IL-6) by blocking NF-κB and p38 kinase in BPA-induced HMC-1 cells.     

Dried chicory root modifies the activity and expression of porcine hepatic CYP3A but not 2C – Effect of in vitro and in vivo exposure

Hepatic cytochrome P450 expression and activity are dependent on many factors, including dietary ingredients. In the present study, we investigated the in vivo and in vitro effect of chicory root on hepatic CYP3A and 2C in male pigs. Chicory feeding increased the expression of CYP3A29 mRNA but not CYP2C33. Correspondingly, CYP3A activity was increased by chicory feeding, while CYP2C activity was not affected. Additionally, the in vitro effect of chicory extract on the CYP3A activity was investigated. It was shown that CYP3A activity in the microsomes from male pigs was inhibited, but this effect was eliminated by pre-incubation. In both male and female pigs the CYP3A activity was increased in the presence of chicory after pre-incubation. Furthermore, gender-related differences in mRNA expression and activity were observed. CYP3A mRNA expression was greater in female pigs; this was not reflected on activity. For CYP2C, no difference in mRNA expression was observed, while CYP2C activity was greater in female pigs. Surprisingly, the expression of the constitutive androstane receptor, pregnane X receptor and aryl hydrocarbon receptor did not differ with feed or gender. In conclusion, chicory root modifies the expression and activity of CYP3A in vivo and in vitro, while CYP2C is not affected.     

Intrinsic apoptosis and NF-κB signaling are potential molecular targets for chemoprevention by black tea polyphenols in HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo

Antiproliferative and apoptosis inducing effects of black tea polyphenols (Polyphenon-B) on HepG2 cells in vitro and in a rat hepatocarcinogenesis model in vivo were investigated. Viability of HepG2 cells was evaluated by the MTT assay, and apoptosis by AO–EB and DAPI staining, cell cycle analysis, and annexin V-PI assay. For the in vivo study, male Sprague–Dawley rats treated with dimethylaminoazobenzene (DAB) (0.06%) were used. The expression of Bcl-2 and NF-κB family members were analyzed by immunoblotting. Administration of Polyphenon-B induced dose-dependent inhibition of growth of HepG2 cells and reduced tumor incidence in DAB administered animals. HepG2 cells also exhibited morphological features characteristic of apoptotic cell death. In addition, administration of Polyphenon-B increased the expression of Bax, tBid, Smac/Diablo, cytochrome C, Apaf-1, caspases, and IκB with PARP cleavage, and decreased the expression of Bcl-2, Bcl-xL, pBad, NF-κB, p-IκB-α, IKKβ and Ub in both HepG2 cells and in DAB-treated animals. These results provide evidence that Polyphenon-B effectively inhibits proliferation and induces apoptosis both in vitro and in vivo by inhibiting NF-κB, and inducing intrinsic apoptosis by modulating the expression of a network of interrelated molecules eventually culminating in caspase-mediated cell death.     

A comparative study of the anticlastogenic effects of chlorophyllin on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz (α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo

Chlorophyllin (CHL), a water soluble derivative of chlorophyll has been shown to have both anticarcinogenic and antigenotoxic properties. We evaluated the protective effects of CHL (25 μM in vitro, 4 and 100 mg/kg. b.w.) on the clastogenic action of two model carcinogens, MNNG and DMBA (25 μM and 2 μM respectively) in vitro on human hepatoma cells (HepG2) and (40 mg and 25 mg/Kg/b.w. respectively) in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with CHL and the carcinogen were carried out in vitro. With MNNG, only simultaneous treatment with CHL was effective in reducing the frequencies of MN, suggesting a direct interaction between CHL and MNNG. A statistically significant reduction in of DMBA induced MN was found by pre-or post treatment with CHL while a reduction (not significant) was observed by simultaneous treatment. In in vivo experiments, CHL pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by MNNG or DMBA. However, increased the toxic effect of DMBA (reduction in percent of PCEs) was accompanied by a reduction in the induced frequencies of MN. CHL was not clastogenic in both in vitro and in vivo tests. It can be concluded that (a) CHL has a protective effect against MNNG and DMBA. This effect is dependent upon the protocol employed in in vitro experiments. In vivo, CHL did not have a protective effect against MNNG and DMBA. A protective effect of CHL against DMBA was evident only at high toxic levels.     

Catechol metabolites of zeranol and 17β-estradiol: A comparative in vitro study on the induction of oxidative DNA damage and methylation by catechol-O-methyltransferase

α-Zearalanol (α-ZAL, zeranol) is a highly estrogenic macrocyclic β-resorcylic acid lactone, which is used as a growth promotor for cattle in various countries. We have recently reported that α-ZAL and its major metabolite zearalanone (ZAN) are hydroxylated at the aromatic ring by microsomes from human liver in vitro, thereby forming two catechol metabolites each. Thus, the oxidative metabolism of α-ZAL and ZAN resembles that of the endogenous steroidal estrogens 17β-estradiol (E2) and estrone (E1), which also give rise to two catechols each. As these catechol metabolites are believed to mediate the carcinogenicity of E2 and E1 by causing oxidative DNA damage and DNA adducts, their methylation by catechol-O-methyltransferase (COMT) is an important inactivation pathway. Here we report that hepatic microsomes from five species generate catechol metabolites of α-ZAL and ZAN, the highest amounts being formed by human liver microsomes, followed by rat, mouse, steer and swine. The microsomal extracts and the individual catechols of α-ZAL, ZAN, E2 and E1 were found to induce oxidative DNA damage, as measured by the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine in a cell-free system. The ranking of pro-oxidant activity was 15-HO-ZAN > 15-HO-α-ZAL ≈ 4-HO-E2/E1 ≈ 2-HO-E2/E1 > 13-HO-ZAN > 13-HO-α-ZAL. With respect to the rate of methylation by human hepatic COMT, the ranking was 2-HO-E2/E1 >> 4-HO-E2/E1 > 15-HO-α-ZAL/ZAN >> 13-HO-α-ZAL/ZAN. Thus, some catechol metabolites of α-ZAL and ZAN are better pro-oxidants and poorer substrates of COMT than the catechols of E2 and E1. These findings warrant further investigations into the genotoxic potential of α-ZAL, which may constitute another biological activity in addition to its well-known estrogenicity.     

Cry1Ac protoxin from Bacillus thuringiensis promotes macrophage activation by upregulating CD80 and CD86 and by inducing IL-6, MCP-1 and TNF-α cytokines

Bacillus thuringiensis Cry1Ac protoxin (pCry1Ac) is a promising mucosal adjuvant, but its action mechanism is unknown. We examined in vivo whether pCry1Ac promotes the activation of macrophages in the peritoneum, spleen and mesenteric lymph nodes or in the lungs and bronchoalveolar lavage after intraperitoneal or intranasal pCry1Ac administration, respectively, in BALB/c mice. pCry1Ac upregulated the costimulatory molecules CD80 and CD86 in these macrophages, but with distinct kinetics. In vitro stimulation of resident macrophages with pCry1Ac upregulated CD80 and CD86 and enhanced the production of the pro-inflammatory cytokines TNF-α, IL-6 and MCP-1. To investigate whether the pCry1Ac-induced activation was mediated through MAPK pathways, we pretreated RAW 264.7 cells with signaling inhibitors of MEK, JNK and p38 MAPKs (PD98059, SP600125 and SB203580, respectively). pCry1Ac-induced upregulation of CD86 and CD80 was partially inhibited by the MEK inhibitor. While LPS-induced upregulation mechanisms of CD80 and CD86 appear to be different; as these were particularly inhibited by MEK and JNK inhibitors, respectively. pCry1Ac-induced IL-6 and MCP-1 production was especially inhibited with the p38 MAPK inhibitor, whereas TNF-α was only slightly inhibited upon treatment with JNK and p38 MAPK inhibitors. Therefore macrophage stimulation with pCry1Ac induced the upregulation of CD80 and CD86, and the production of IL-6, TNF-α and MCP-1, possibly, through the MEK and p38 MAPK pathways. It also promoted the nuclear translocation of NF-κB p50 and p65, the upregulation of MHC-II, and the activation of T CD4+ cells. These results suggest that pCry1Ac induced macrophage activation through mechanisms which differ partially from the LPS-induced.     

Anti-rheumatic activity of pseudoephedrine (a substituted phenethylamine) in complete Freund’s adjuvant-induced arthritic rats by down regulating IL-1β, IL-6 and TNF-α as well as upregulating IL-4 and IL-10

Inflammopharmacology - Pseudoephedrine (substituted phenethylamine) is well known as psychotic and bronchodilator. Numerous studies on phenethylamine derivatives indicated that these agents have...     

Effect of Diacerein on HOTAIR/IL-6/STAT3, Wnt/β-Catenin and TLR-4/NF-κB/TNF-α axes in colon carcinogenesis

Colorectal cancer (CRC) is a common malignancy with high mortality and poor prognosis. Diacerein (DIA) is an anti-inflammatory used for treatment of osteoarthritis. We delineated some underlying molecular mechanisms of DIA’s anti-carcinogenic effect in CRC using in vivo and in vitro models. Human Caco-2 cells were treated with DIA followed by MTT and Annexin V assays and CRC was experimentally induced using 1,2-dimethylhydrazine. DIA (50 mg/kg/day, orally) was administrated for 8 weeks. The MTT assay confirmed cytotoxic effect of DIA in vitro and Annexin V confirmed its apoptotic effect. DIA resulted in regression of tumour lesions with reduced colonic TLR4, NF-κB and TNF-α protein levels and down-regulated VEGF expression, confirming anti-angiogenic impact. DIA triggered caspase-3 expression and regulated Wnt/β-Catenin pathway, by apparently interrupting the IL-6/STAT3/ lncRNA HOTAIR axis. In conclusion, DIA disrupted IL-6/STAT3/ lncRNA HOTAIR axis which could offer an effective therapeutic strategy for the management of CRC.     

A crucial role for TNF-α in mediating neutrophil influx induced by endogenously generated or exogenous chemokines,KC/CXCL1 and LIX/CXCL5

Background and purpose:

Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP-2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice.

Experimental approach:

Neutrophil recruitment was assessed in immunized mice challenged with methylated bovine serum albumin, KC/CXCL1, LIX/CXCL5 or tumour necrosis factor (TNF)-α. Cytokine and chemokine levels were determined in peritoneal exudates and in supernatants of macrophages and mast cells by elisa. CXCR2 and intercellular adhesion molecule 1 (ICAM-1) expression was determined using immunohistochemistry and confocal microscopy.

Key results:

Antigen challenge induced dose- and time-dependent neutrophil recruitment and production of KC/CXCL1, LIX/CXCL5 and TNF-α, but not MIP-2/CXCL2, in peritoneal exudates. Neutrophil recruitment was inhibited by treatment with reparixin (CXCR1/2 antagonist), anti-KC/CXCL1, anti-LIX/CXCL5 or anti-TNF-α antibodies and in tumour necrosis factor receptor 1-deficient mice. Intraperitoneal injection of KC/CXCL1 and LIX/CXCL5 induced dose- and time-dependent neutrophil recruitment and TNF-α production, which were inhibited by reparixin or anti-TNF-α treatment. Macrophages and mast cells expressed CXCR2 receptors. Increased macrophage numbers enhanced, while cromolyn sodium (mast cell stabilizer) diminished, LIX/CXCL5-induced neutrophil recruitment. Macrophages and mast cells from immunized mice produced TNF-α upon LIX/CXCL5 stimulation. Methylated bovine serum albumin induced expression of ICAM-1 on mesenteric vascular endothelium, which was inhibited by anti-TNF-α or anti-LIX/CXCL5.

Conclusion and implications:

Following antigen challenge, CXCR2 ligands are produced and act on macrophages and mast cells triggering the production of TNF-α, which synergistically contribute to neutrophil recruitment through induction of the expression of ICAM-1.