Semiquinone fraction isolated from Bacillus sp. INM‐1 protects hepatic tissues against γ‐radiation induced toxicity

Present study was focused on evaluation of a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM‐1 for its ability against γ radiation induced oxidative stress in irradiated mice. Animals were divided into four group, i.e., (i) untreated control mice; (ii) SQGD treated (50 mg/kg b. wt. i.p.) mice; (iii) irradiated (10 Gy) mice; and (iv) irradiated mice which were pre‐treated (?2 h) with SQGD (50 mg/kg b. wt. i.p.). Following treatment, liver homogenates of the treated mice were subjected to endogenous antioxidant enzymes estimation. Result indicated that SQGD pre‐treatment, significantly (P < 0.05) induced superoxide dismutase (SOD) (19.84 ± 2.18% at 72 h), catalase (CAT) (26.47 ± 3.11% at 12 h), glutathione (33.81 ± 1.99% at 24 h), and glutathione‐S‐transferase (24.40 ± 2.65% at 6 h) activities in the liver of mice as compared with untreated control. Significant (P < 0.05) induction in SOD (50.04 ± 5.59% at 12 h), CAT (62.22 ± 7.50 at 72 h), glutathione (42.92 ± 2.28% at 24 h), and glutathione‐S‐transferase (46.65 ± 3.25 at 12 h) was observed in irradiated mice which were pre‐treated with SQGD compared with only irradiated mice. Further, significant induction in ABTS⁺ radicals (directly proportional to decrease mM Trolox equivalent) was observed in liver homogenate of H₂O₂ treated mice which were found to be significantly inhibited in H₂O₂ treated mice pre‐treated with SQGD. Thus, it can be concluded that SQGD treatment neutralizes oxidative stress caused by irradiation not only by enhancing endogenous antioxidant enzymes but also by improving total antioxidant status of cellular system and thus cumulative effect of the phenomenon may contributes to radioprotection. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1471–1478, 2014.     

In vitro stimulatory effect of N‐acetyl tryptophan‐glucopyranoside against gamma radiation induced immunosuppression

Radiation‐induced manifestations like free radical burst, oxidative damage and apoptosis leading to cell death. In present study, N‐acetyl tryptophan glucopyranoside (NATG) was assessed for its immune‐radioprotective activities using J774A.1 cells. Clonogenic cell survival, cell cycle progression and cytokines i.e. IFN‐γ, TNF‐α, IL‐2, IL‐10, IL‐12, IL‐13 and IL‐17A expression were evaluated in irradiated and NATG pretreated cells using clonogenic formation ability, flow cytometry and ELISA assay. Results indicated that 0.25μg/ml NATG exhibited maximum radioprotection against gamma‐radiation (2Gy) without intervening in cell cycle progression. NATG pretreated (−2 h) plus irradiated cells showed significant elevation in IFN‐γ (∼38.2%), IL‐17A (∼53.7%) and IL‐12 (∼58.8%) expression as compared to only irradiated cells. Conversely, significant decrease in TNF‐α (∼21.6%), IL‐10 (∼31.2%), IL‐2 (∼23.7%) and IL‐13 expression (∼17.8%) were observed in NATG pretreated plus irradiated cells as compared to irradiated cells. Conclusively, NATG pretreatment to irradiated J774A.1 cells, stimulate Th₁ while diminish Th₂ cytokines that contributes to radioprotection.     

A semiquinone glucoside derivative provides protection to male reproductive system of the mice against gamma radiation toxicity

Present investigation was carried out to evaluate the radioprotective efficacy of a novel Semiquinone glucoside derivative (SQGD), isolated from Bacillus sp. INM‐1, in the male reproductive system of BALB/c mice. Animals were administered 50 mg/kg b.wt. (i.p.) SQGD 2 h before whole body γ‐irradiation (10 Gy). Radiation‐induced cellular toxicity and its modulation by SQGD pretreatment was evaluated in the mice testes by quantitative histological and protein expression analysis. SQGD pretreatment protects irradiated mice from radiation‐induced testicular atrophy and germ cells degeneration, which may lead to emptiness of seminiferous tubules. Significant decrease in P53 and P21^{(Cip/WAF‐1)} expression was observed in the irradiated mice pretreated (2 h) by SQGD at 6 h compared with only irradiated mice. However, contrary to P53, expressions of P21 at latter time, that is, 24–72 h was found to be increased significantly in the irradiated mice pretreated by SQGD. Significant increase in the intact PARP‐1 protein expression were observed in the testes of the mice pretreated by SQGD 2 h before irradiation at 24–72 h compared with the only irradiated mice, whereas significant increase in PARP‐1 cleaved fragment was noticed at 24 h. Similarly, significant increase in NF‐kB and BCL‐2/BAX expressions ratio was noticed in SQGD‐treated mice (± irradiation) compared with irradiated mice, suggested a role of SQGD in the activation of prosurvival signaling in the testicular germinal cells population of the irradiated mice and thus contributed to protection against lethal γ‐irradiation. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 558–567, 2014.     

Anti‐viral activity of jatrophone against RSV‐induced respiratory infection via increase in interferon‐γ generating dendritic cells

Respiratory syncytial virus (RSV), a member of Paramyxoviridae family is responsible for bronchiolitis and pneumonia. The present study investigated anti‐viral and anti‐inflammatory activities of jatrophone against RSV‐infection in pulmonary epithelial cells in vitro and in mice model in vivo. The changes in viabilities of RSV infected cells by jatrophone treatment were determined by MTT assay. The fluorescence associated with production of ROS was evaluated by fluorescence microscopy using H2DCFDA dye. The IFN‐γ secreting cells were detected in mice BALF by stimulation with phorbol myristate acetate and ionomycin. The reduction of BEAS‐2B cell viability by RSV was alleviated on treatment with jatrophone in dose based manner. The cytopathogenic changes by RSV infection were prevented and viral growth inhibited by jatrophone in BEAS‐2B cells. Jatrophone treatment significantly alleviated RSV mediated overproduction of IL‐6/‐8 and suppressed ROS generation in the cells. The pulmonary viral titers were found to be markedly lower in mice treated with jatrophone relative to untreated group. The jatrophone treated mice also showed reduced IL‐4/‐5/‐13 levels and elevated IFN‐γ level in BALF relative to untreated RSV infected mice. Flow cytometry revealed elevated count of IFN‐γ generating cells in RSV infected mice on treatment with jatrophone. Thus jatrophone inhibits viral growth and oxidative damage by RSV in pulmonary epithelial cells. In RSV infected mice jatrophone increased immunity for viral infection by modulating cell phenotype for promotion of anti‐viral IFN‐γ. Thus jatrophone acts as potential anti‐viral compound and may be developed for treatment of respiratory treat infection.     

β-1,3-Glucan given orally modulates immunomyelopoietic activity and enhances the resistance of tumour-bearing mice

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Emodin attenuates acute rejection of liver allografts by inhibiting hepatocellular apoptosis and modulating the Th1/Th2 balance in rats

1. In the present study, we investigated the immunosuppressive effects and mechanisms of action of emodin on acute graft rejection following liver transplantation in a rat model of orthotopic liver transplantation. 2. Rats were divided into three groups: Group A, syngenic control (Brown Norway‐to‐Brown Norway); Group B, acute rejection group (Lewis‐to‐Brown Norway); and Group C, emodin‐treated group (Lewis‐to‐Brown Norway treated with 50 mg/kg emodin, 50 mg/kg.d, injected intraperitoneally once a day from days 1 to 5 posttransplantation). The survival time of the recipients in each group was recorded. Histopathological changes in the liver, hepatocellular apoptosis, serum concentrations of interleukin (IL)‐2, interferon (IFN)‐γ and IL‐4 and their expression in liver tissue were determined. 3. Emodin treatment prolonged liver allograft survival time from 10.9 days in Group B to 25.6 days in Group C. The rejection activity index (calculated according to the Banff Schema) in Groups A, B and C was 1.29 ± 0.47, 7.58 ± 0.85 and 4.72 ± 0.79, respectively (P < 0.01 for Groups A and B vs Group C), whereas the apoptosis index in the three groups was 15.51 ± 1.47, 39.50 ± 1.65 and 16.72 ± 1.73, respectively (P < 0.01 for Groups A and C vs Group B). Serum levels of IL‐2 and IFN‐γ were higher, whereas levels of IL‐4 were lower, in the acute rejection group (Group B) than in the emodin‐treated group (Group C; P < 0.05). Changes in the expression of these cytokines in transplanted liver tissue were consistent with changes in serum concentrations. 4. In conclusion, emodin effectively suppresses acute graft rejection in vivo to prolong the survival of recipient rats. The mechanism underlying this effect may be associated with the prevention of hepatocyte apoptosis and with a changing in the balance of Th1/Th2 cytokines towards Th2.     

Immunological Response to (1,4)‐α‐d‐Glucan in the Lung and Spleen of Endotoxin‐Stimulated Juvenile Rats

Abstract: We investigated the effects of (1,4)‐α‐D‐glucan (α‐DG), a novel immune stimulatory drug from Tinospora cordifolia, on the concentration of pro‐ and anti‐inflammatory cytokines (interleukin [IL]‐1β, IL‐6, tumour necrosis factor‐α [TNF‐α], γ‐interferon [IFN‐γ] and IL‐10) in the lung and spleen of endotoxin‐stimulated juvenile rats. Experimental groups (n = 16/group) included controls with an intraperitoneal injection of saline, endotoxaemic rats with a non‐lethal dose of 10 mg/kg Escherichia coli endotoxin, and endotoxaemic rats treated with two doses of 10 mg/kg α‐DG, intraperitoneally, 2 and 4 hr after endotoxin injection. At 24 hr of treatment, rats were euthanized and lungs and spleen were removed for cytokines determination and lung injury. Endotoxaemia increased IL‐1β concentration by fivefold in both organs, while creating a moderate pulmonary hypercellularity (demonstrated by about 11% increase in the alveolar‐septal thickening and 11% decrease in the alveolar‐interstitial space ratio). In the lung, α‐DG treatment reduced concentrations of IL‐1β by 30% (p > 0.05), IL‐6 by 43% (p < 0.01), IFN‐γ by 46% (p < 0.01) and the anti‐inflammatory cytokine, IL‐10, by 31% (p > 0.05) compared to endotoxaemia. In the spleen, α‐DG treatment decreased the ratio of IL‐1β to IL‐10 by 55% (p < 0.05), demonstrating an anti‐inflammatory trend. These data suggest that α‐DG differentially modulates cytokine response in the lung and spleen and modifies the pro‐ and anti‐inflammatory balance during an early period of endotoxaemia in juvenile rats.     

Low‐dose metronomic chemotherapy with cisplatin enhanced immunity in a murine model of ectopic cervical cancer

Previous researchers have claimed that metronomic low‐dose/dense chemotherapy can enhance the therapeutic effectiveness of cisplatin treatment in the control of cancer. Therefore, the aim of this study was to explore the effectiveness of metronomic drug delivery with regards to its effects on adaptive immunity in a murine model of ectopic cervical cancer. The effectiveness of long‐term low‐dose/dense cisplatin treatment in HPV E7‐expressing TC‐1 cells was evaluated via morphological observations. Tumour mass and survival curves were used to determine the antitumour effect against E7‐expressing tumours. After experimental mice had been treated with low‐dose/dense cisplatin therapy, flow cytometry was used to measure the expression of MHC class I surface antigens on cultured TC‐1 cells. Splenocytes expressing both interferon (IFN)‐γ and CD8 responsible for E7 antigens and the T_reg population were also quantified using flow cytometry. The results indicate that in vivo treatment with metronomic cisplatin suppresses the growth of cultured TC‐1 cells. An increase was also observed in the number of splenocytes expressing both IFN‐γ and CD8 responsible for E7 antigens and the T_reg population. These results support previous reports that metronomic low‐dose/dense cisplatin chemotherapy is an effective treatment against ectopic cervical cancer with E7‐expression.     

Brominated flame retardants stimulate mouse immune cells in vitro

Brominated flame retardants (BFRs) are widely used in consumer products. Their toxicological effects as endocrine disruptors have been partly examined. However, their immunological effects have not been elucidated. To evaluate the effects of BFRs on immune responses, we investigated whether BFRs affect phenotypes and the function of immune cells in vitro. Here we examined the commercial pentabromodiphenyl ether mixture (DE‐71), octabromodiphenyl ether mixture (DE‐79), decabromodiphenyl ether mixture (DE‐83R), hexabromocyclododecane (HBCD) and tetrabromobisphenol A (TBBPA). Splenocytes and bone marrow (BM) cells were prepared from atopic prone NC/Nga mice. Splenocytes were exposed to each BFR for 24 h. BM cells were cultured with granulocyte macrophage‐colony stimulating factor (GM‐CSF) for 8 days and BM‐derived dendritic cells (BMDCs) were exposed to each BFR for 24 h. In another experiment, BM cells were cultured with GM‐CSF in the presence of each BFR for 6 days during BMDC differentiation. After exposure, cell surface molecule expression and cytokine production were investigated. Each BFR increased MHC class II and CD86 expression and interleukin (IL)‐4 production in splenocytes. DE‐71, HBCD and TBBPA increased T cell receptor (TCR) expression in splenocytes. In both experiments, all BFRs except TBBPA increased DEC205 expression in BMDCs. BMDCs that differentiated in the presence of HBCD showed enhanced MHC class II, CD80, CD86 and CD11c expression. The results demonstrate that some BFRs may stimulate immune cells. BFRs can induce or enhance immune/allergic responses by increasing antigen presentation‐related molecule expression and IL‐4 production. Copyright © 2012 John Wiley & Sons, Ltd.     

Dehydroxymethylepoxyquinomicin,a novel nuclear factor‐κB inhibitor,prevents inflammatory injury induced by interferon‐γ and histamine in NCTC 2544 keratinocytes

1. The novel nuclear factor (NF)‐κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is a derivative of the antibiotic epoxyquinomicin C from Amycolatopsis sp. that has been found to inhibit tumour necrosis factor (TNF)‐α‐induced activation of NF‐κB by suppressing nuclear translocation of NF‐κB. The aim of the present study was to determine the effects of DHMEQ on interferon (IFN)‐γ‐ and histamine‐activated NCTC 2544 keratinocytes. 2. Keratinocytes were stimulated or not with 200 U/mL IFN‐γ and 10^?4 mol/L histamine in the absence or presence of different concentrations of DHMEQ (1, 5 and 10 μg/mL) or hydrocortisone (10^?5 mol/L), which was used as a reference anti‐inflammatory drug. After 48 h, each sample was tested for the presence of intercellular adhesion molecule (ICAM)‐1 by western blot analysis, as well as for the release of monocyte chemoattractant protein (MCP)‐1, RANTES and interleukin (IL)‐8 using specific sandwich ELISAs. To verify the effect of DHMEQ on cell viability of non‐stimulated NCTC 2544 keratinocytes, the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay was used. 3. The results showed that 10 μg/mL DHMEQ potently inhibited ICAM‐1 production (by 50%), as well as the release of MCP‐1 (to 25% of control), RANTES (to 5% of control) and IL‐8 (to 2% of control). The results of the MTT assay indicated that DHMEQ has no effect on cell viability. 4. In conclusion, DHMEQ inhibits the IFN‐γ‐ and histamine‐induced activation of the keratinocyte cell line NCTC 2544. The anti‐inflammatory effects of DHMEQ could be exploited by applying the drug topically alone or in combination with sub‐toxic concentrations of anti‐inflammatory drugs to producer a synergistic effect.     

Physiological Effects of a Novel Immune Stimulator Drug, (1,4)‐α‐d‐Glucan,in Rats

Abstract: The (1,4)‐α‐d ‐glucan (α‐d ‐glucan), derived from medicinal plant, Tinospora cordifolia, activates human lymphocytes with downstream synthesis of the pro‐ and anti‐inflammatory cytokines, in vitro. We investigated physiological and immunological effects of a low and a high dose of α‐d ‐glucan (0.5 and 10 mg/kg), in vivo, testing the hypothesis that intravenous administration of α‐d ‐glucan does not affect haemodynamic, respiratory, haematological, and immune responses in normal rats. Male rats (300–400 g) were anaesthetized, tracheostomized, and catheterized in one femoral artery and vein. The mean arterial blood pressure and heart rate were continuously recorded. The baselines for gas exchange, differential blood cell count, and plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ were determined. Rats were then randomly assigned to controls (n = 7), a low dose (0.5 mg/kg; n = 10), and a high dose (10 mg/kg; n = 7) of α‐d ‐glucan for a six 6 hr study period. Gas exchange, differential cell count, plasma concentration of TNF‐α, IL‐1β, IL‐4, IL‐6, and IFN‐γ, and mean arterial blood pressure values remained within physiological range. Intravenous administration of 10 mg/kg α‐d ‐glucan created tachycardia, associated with hyperventilation, and significant reductions in the blood haemoglobin and haematocrit concentrations. We suggest that these in vivo effects of α‐d ‐glucan should be considered for future clinical and/or experimental trials.     

Semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1 protects against gamma radiation-induced oxidative stress

In the present study, radioprotective potential of Semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 was evaluated. γ-Radiation induced protein carbonylation, plasmid DNA damage, enzyme functional impairment, lipid peroxidation, HO radicals generation and their protection by SQGD was assessed. As a result of SQGD treatment, significant inhibition (p < 0.05) in protein carbonylation was observed with BSA. SQGD treatment was found to restore supercoiled (∼70 ± 3.21%) form of irradiated plasmid DNA against γ-irradiation. SQGD protects enzymes (EcoR1 and BamH1) against radiation-induced dysfunctioning. SQGD significantly inhibited (p < 0.05) lipid peroxidation in liposomes, brain and liver homogenate. Higher HO^• radicals-averting activity of SQGD was observed in the serum and liver homogenate of C57BL/6 mice against H₂O₂-induced oxidative stress. In conclusion, SQGD demonstrates excellent radical-scavenging activity towards bio-macromolecules in irradiated environment and can be developed as an ideal radioprotector against radiation-induced oxidative stress in future.     

Comparative effects of 1α‐hydroxyvitamin D3 and 1,25‐dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(‐/‐) mice

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P‐gp expression. The present study compared the equimolar efficacies of 1α‐hydroxyvitamin D₃ [1α(OH)D₃] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half‐life that is converted to 1,25(OH)₂D₃, and 1,25(OH)₂D₃ on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)‐directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(‐/‐)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D₃ and 1,25(OH)₂D₃, 1α(OH)D₃ treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(‐/‐) mice compared to 1,25(OH)₂D₃ treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P‐gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D₃ treatment. The higher activity of 1α(OH)D₃ was explained by its rapid conversion to 1,25(OH)₂D₃ in tissue sites, furnishing higher plasma and tissue 1,25(OH)₂D₃ levels compared to following 1,25(OH)₂D₃‐treatment. In conclusion, 1α(OH)D₃ exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)₂D₃ in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

MiR‐146a regulates PM1‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells

Exposure to particulate matter (PM) leads to kinds of cardiopulmonary diseases, such as asthma, COPD, arrhythmias, lung cancer, etc., which are related to PM‐induced inflammation. We have found that PM_2.5 (aerodynamics diameter <2.5 µm) exposure induces inflammatory response both in vivo and in vitro. Since the toxicity of PM is tightly associated with its size and components, PM₁ (aerodynamics diameter <1.0 µm) is supposed to be more toxic than PM_2.5. However, the mechanism of PM₁‐induced inflammation is not clear. Recently, emerging evidences prove that microRNAs play a vital role in regulating inflammation. Therefore, we studied the regulation of miR‐146a in PM₁‐induced inflammation in human lung bronchial epithelial BEAS‐2B cells. The results show that PM₁ induces the increase of IL‐6 and IL‐8 in BEAS‐2B cells and up‐regulates the miR‐146a expression by activating NF‐κB signaling pathway. Overexpressed miR‐146a prevents the nuclear translocation of p65 through inhibiting the IRAK1/TRAF6 expression, and downregulates the expression of IL‐6 and IL‐8. Taken together, these results demonstrate that miR‐146a can negatively feedback regulate PM₁‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells.