Radiation protection of HepG2 cells by Podophyllum hexandrum Royale

Radioprotection by an aqueous extract of Podophyllum hexandrum (RP-1) was investigated in HepG2 cells by evaluating colony forming efficacy (CFE), redox status of mitochondria, reactive oxygen species (ROS), generation of nitric oxide (NO), peroxidation of lipids and intracellular glutathione. Lower concentrations of RP-1 (0.1 and 1 g/ml) rendered maximum radioprotection when administered 1 or 2 h before irradiation. Higher concentrations (5 and 10 g/ml) however were less effective when administered 1 or 2 h before irradiation, but were more effective with increased time intervals (4 or 8 h) between RP-1 administration and irradiation. RP-1 pre-treatment also significantly inhibited radiation-induced MTT reduction in a concentration and time-dependent manner by decreasing gamma radiation-induced leakage of electrons from electron transport chain. Pre-irradiation administration of RP-1 significantly reduced both ROS and NO generation and enhanced glutathione levels, thereby inhibiting lipid peroxidation.     

GA3 induced flowering in Podophyllum hexandrum Royle: A rare alpine medicinal herb

Podophyllum hexandrum Royle, an important alpine herb, is the source of highly valued podophyllotoxin. The effect of some plant growth substances (GA₃, BAP & ABA), uniconazole (an inhibitor of GA biosynthesis), and a combination of GA₃ and uniconazole were examined in respect to influence on sprouting in rhizomes of P. hexandrum and on induction of flowering at a lower altitude. Amongst the various chemicals tested, GA₃ had a marked effect resulting in uniform sprouting and also induced flowering in about half of the treated rhizomes. While BAP also promoted early sprouting, delayed sprouting was seen in rhizomes treated with ABA. Uniconazole treatment, either alone or with GA₃ was found to inhibit flowering and also resulted in reduced plant height. GA₃ treatment of rhizomes from plants that was maintained for up to 30 months at a lower altitude also induced flowering thus replacing the normal chilling requirement of plants. These results suggest that treatment of GA₃ could be effectively used for inducing uniform sprouting and flowering in rhizomes of P. hexandrum grown at lower altitudes.     

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

Temperature-dependent growth and emergence of functional leaves: an adaptive mechanism in the seedlings of the western Himalayan plant Podophyllum hexandrum

As an adaptive mechanism, hypocotyl dormancy delays emergence of functional leaf until favorable season of growth in Podophyllum hexandrum, an endangered medicinal plant of the western Himalayas. However, upon exposure of the freshly germinated seedlings to favorable temperature (25°C), functional leaves emerged within 20 days. Therefore, we examined regulation mechanisms of growth and development of this alpine plant by temperature under laboratory conditions. The seedlings were exposed to (1) 25°C (temperature prevailing at the time of maximum vegetative growth), (2) 4°C (mean temperature at the onset of winter in its natural habitat), and (3) 10°C (an intermediate temperature). Slackened growth at 4°C was followed by senescence of aerial parts and quiescence of roots and predetermined leaf primordia. Rapid development of leaf primordia at 25°C was associated with increased starch hydrolysis. This was evident from higher α-amylase activity and reducing sugars. These parameters decreased on sudden exposure to 4°C. In contrast, the roots (perennating organs) showed a slight increase (1.36-fold) in α-amylase activity. Growth and development in seedlings growing at 10°C (temperature less adverse than 4°C) were comparatively faster. The content of reducing sugars and α-amylase activity were also higher in all the seedling parts at 10°C as compared to 4°C. This indicated larger requirements for sugar by the seedlings at 10°C. Irrespective of temperature, maximum changes in nitrate and nitrate reductase occurred during the initial 10 days, i.e., when the readily available form of sugars (reducing sugar) was highest. This indicated that a temperature-dependent availability of carbon, but not temperature itself, was an important regulator of uptake and reduction of nitrogen. IHBT Publication number 508a.     

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 g/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 g/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.     

DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences (p<0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells.     

The immunomodulation potential of the synthetic derivatives of benzothiazoles: Implications in immune system disorders through in vitro and in silico studies

Benzothiazole and its natural or synthetic derivatives have been used as precursors for several pharmacological agents for neuroprotective, anti-bacterial, and anti-allergic activities. The objecctive of the present study was to evaluate effects of benzothiazole analogs (compounds 1–26) for their immunomodulatory activities. Eight compounds (2, 4, 5, 8–10, 12, and 18) showed potent inhibitory activity on PHA-activated peripheral blood mononuclear cells (PBMCs) with IC₅₀ ranging from 3.7 to 11.9 μM compared to that of the standard drug, prednisolone <1.5 μM. Some compounds (2, 4, 8, and 18) were also found to have potent inhibitory activities on the production of IL-2 on PHA/PMA-stimulated PBMCs with IC₅₀ values ranging between <4.0 and 12.8 μM. The binding interaction of these compounds was performed through silico molecular docking. Compounds 2, 8, 9, and 10 significantly suppressed oxidative burst ROS production in phagocytes with IC₅₀ values between <4.0 and 15.2 μM. The lipopolysaccharide (LPS)-induced nitrites in murine macrophages cell line J774 were found to be inhibited by compounds 4, 8, 9, and 18 at a concentration of 25 μg/mL by 56%, 91%, 58%, and 78%, respectively. Furthermore, compounds 5, 8, 12, and 18 showed significant (P < 0.05) suppressive activity on Th-2 cytokine, interleukin 4 (IL-4) with an IC₅₀ range of <4.0 to 40.3 μM. Interestingly compound 4 has shown a selective inhibitory activity on IL-2 and T cell proliferation (naïve T cell proliferation stage) rather than on IL-4 cytokine, while compound 12 displayed an interference with T-cell proliferation and IL-4 generation. Moreover compound 8 and 18 exert non-selective inhibition on both IL-2 and IL-4 cytokines, indicating a better interference with stage leading to humoral immune response and hence possible application in autoimmune diseases.     

Inhibitory effects of Azadirachta indica on DMBA-induced skin carcinogenesis in Balb/c mice

Male Balb/c mice were divided into four groups on the basis of their respective treatments wherein mice of Group I served as controls. For induction of skin tumors, mice of Group II and IV were injected sub-cutaneously with 7,12-dimethylbenz(a)anthracene (DMBA). Mice of Group III and IV were administered aqueous Azadirachta indica leaf extract (AAILE) thrice a week throughout the experiment. After 14 weeks of the first DMBA injection, Group II and IV mice developed tumors. In the tumor-bearing mice that received AAILE (Group IV), a significant reduction in mean tumor burden and tumor volume was observed. The tumors were confirmed to be papillomas and interestingly, the extent of hyper-chromatia was observed to be much more in skin tumors of Group II mice vis a vis the mice receiving AAILE. An increase in the extent of lipid peroxidation was observed in tumorous tissue of Group IV when compared to that of Group II mice. Glutathione (GSH) content and the activities of GSH-based antioxidant enzymes viz. glutathione peroxidase (GPx) and glutathione reductase (GR) increased significantly in the skin tissues of all the groups of mice when compared to control counterparts. Catalase activity was found to decrease significantly in the skin of mice, which received AAILE treatment only (Group III). Activity of super-oxide dismutase (SOD) decreased significantly in all the tumorous tissues (Group II and IV mice). In light of the above observations, the role of AAILE in inhibition of DMBA-induced skin carcinogenesis is discussed in the present study.     

Influence of Th1/Th2 Cytokines and Nitric Oxide in Murine Systemic Infection Induced by Sporothrix schenckii

In an attempt to elucidate the effects of Sporothrix schenckii infection on the immune response, our laboratory has developed a murine model of disseminated sporotrichosis. Helper T cells can be further subdivided into Th1 and Th2 phenotypes. The differentiation of two subsets of T lymphocytes is driven by IL-12 and IL-4 cytokines, respectively. Th1 cells produce IFN-γ that activate macrophages and promote cell-mediated immunity. In addition, we found low levels of iNOS and NO production in the initial (1st and 2nd weeks) and final (9th and 10th weeks) periods of the infection, in contrast with the period of week 4 to 7 of elevated values. The determination of IFN-γ and IL-12 are in agreement with NO/iNOS detection, showing the presence of cellular immune response throughout the infectious process. However, the production of IL-4 shows an increase in levels after the 5th and 6th weeks suggesting a participation of Th2 response in this period as well. Regarding these results, the study demonstrated that in experimental sporotrichosis infection the cellular immune response participated throughout the period analyzed as a nitric oxide dependent mechanism. In contrast, the presence of Th2 response began in the 5th week, suggesting the participation of humoral immune response in advanced stages of sporotrichosis.     

Cytochrome c oxidase activity in T/t 6 (balanced lethal) mutant mice

R-1 (1450g) and R-2 (25,000g) liver fractions from T/t ⁶ and B6CBAF1 hybrid mice were analyzed for their protein content, mitochondria concentrations, and activities of three respiratory-chain enzymes of the mitochondrial inner membrane: cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, E.C. 1.9.3.1), -glycerophosphate dehydrogenase [l-glycerol-3-phosphate: (acceptor) oxidoreductase, E.C. 1.1.99.5], and succinate-cytochrome c reductase. Only cytochrome c oxidase activity, calculated as units per 1010 mitochondria, was significantly lower in both R-1 and R-2 fractions of T/t ⁶ mice. Cytochrome c oxidase activity varied greatly among T/t ⁶ mice, as did their liver mitochondria concentrations and body weights. Cytochrome c oxidase activity in the R-1 fraction of T/t ⁶ mice, calculated as units per 1010 mitochondria per gram of body weight, averaged about 40% lower than in B6CBAF1 mice. -Glycerophosphate dehydrogenase activity was often elevated in T/t ⁶ mice, particularly in the R-2 fraction. The T/t locus, a complex genetic locus on chromosome 17, may contain genes important to the function and biogenesis of mitochondria.This investigation was supported by institutional funds from the Jackson Laboratory and by an allocation from NIH Biomedical Research Support Grant (RR-05545). The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Pharmacological evaluation of anti-leishmanial activity by in vivo nitric oxide modulation in Balb/c mice infected with Leishmania major MRHO/IR/75/ER: an Iranian strain of cutaneous leishmaniasis

Cutaneous leishmaniasis with a variation in its clinical signs is still one of the health problems in the world, region and Iran. Immune responses against leishmania consist of cytokines, immune cells and mediators. Macrophages participate actively in the inflammatory response by releasing chemokines and mediators including nitric oxide (NO) and reactive oxygen and nitrogen intermediates. This study investigates whether NO had anti-leishmanial effects and/or mediated pathology in mice infected with Leishmania major MRHO/IR/75/ER (IR/75). NO inducer lipopolysaccharide (LPS) and NO donor S-nitrosoglutathione (SNOG) were used for their ability to increase RNI and to modify leishmania infection in susceptible Balb/c mice, in order to evaluate the effects of NO production on size and lesion macroscopy, delay of lesion formation and proliferation of amastigotes inside macrophages. Liver, spleen and lymph nodes were also studied as target organs to detect amastigotes. In addition to plasma, liver and spleen suspensions were investigated for NO induction by using Griess microassay. Statistical analysis of data revealed an association between increases in NO level with the pathology of disease in Balb/c mice infected with L. major IR/75. The survival of leishmania parasite inside the macrophages and its proliferation was affected by LPS and SNOG-treatments. An inconsistent relationship was evident between the NO modulation and pathological changes in treated Balb/c mice infected with L. major IR/75.     

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice

Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210–239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.     

Leishmania chagasi: effect of the iron deficiency on the infection in BALB/c mice

Iron deficiency and visceral leishmaniasis are serious problems of public health. The aim of this study was to evaluate the effect of iron deficiency, induced by the iron chelator desferrioxamine, on the course of the infection by Leishmania chagasi in BALB/c mice. Our data show that the iron chelator caused significant reduction in hemoglobin concentration of treated mice and reduction in parasite load in spleen and liver. Significant differences were not observed in the production of IFN-gamma and IL-4 among the experimental groups. In conclusion, the data reported in this paper suggest that iron deficiency may favor the host. If there is not enough iron available to the parasite, its multiplication may be reduced and infection attenuated.     

Fumonisin B1 alters sphingolipid metabolism and immune function in BALB/c mice: Immunological responses to fumonisin B1

Fumonisins have been reported to have diverse effects on animals, including immunosuppression in chickens and feeder calves; therefore, the effects of fumonisin B₁ (FB₁) on immune function in BALB/c mice was investigated. When administered i.p. with sheep red blood cells (SRBC), 5 to 100 µg of FB₁ reduced the number of plaque-forming cells (PFC) produced against SRBC; however, when administered daily, 1 to 50 µg of FB₁ caused a 4 to 12-fold increase in the number of PFC after SRBC injection. Therefore, FB₁ is not only immunosuppressive; but also, immunostimulatory. To test the possibility that there may have been an immune response to FB₁ as an antigen, FB₁ was injected into mice and the number of splenic cells forming rosettes on FB₁-treated SRBC was determined. There were dose-dependent increases in the antigen-binding cells, with up to 4.9- and 4.6-fold increases, respectively, upon primary and secondary immunization. FB₁-binding immunoglobulins could be detected in sera from some treated mice, but this response was not obtained in every experiment. In summary, these results show that FB₁ has diverse effects on the immune system, causing both stimulation and suppression of the response to foreign antigens, and apparently inducing an antigenic response to FB₁.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - FB₁ fumonisin B₁ - FCS fetal calf serum - PBS phosphate buffered saline - PFC plaque forming cells - RFC rosette forming cells - SRBC sheep red blood cells     

Sequence analysis of the ly49 cluster in C57BL/6 mice: a rapidly evolving multigene family in the immune system

The cytotoxic activity of murine natural killer cells is controlled in part through the action of genes belonging to the Ly49 family. Members of this multigene family are found in a region on mouse chromosome 6 termed the natural killer gene complex. Using data available through public databases, we performed sequence analysis of a 620-kb region in C57Bl/6 (B6) mice that contains the Ly49 genes. The contiguous genomic sequence has allowed us to describe the complete B6 Ly49 gene repertoire, which includes two recently described genes as well as three partial genes. We have shown that the genes in the cluster have evolved through a series of large duplication events involving units of one or more genes and we have attempted to characterize the nature of the duplication end points. Finally, we have used information regarding gene sequence relationships and insertion of repetitive elements to construct a model for the evolution of the gene cluster. Our study illustrates that the Ly49 cluster represents an example of a rapidly evolving gene family, and continued analysis of this region in other strains will undoubtedly provide further insight into mechanisms for generating genomic diversity.     

Trypanosoma cruzi: Effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice

Leukotrienes are important mediators of inflammatory responses. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide (NO) and iNOS expression in cardiac tissue of mice infected with Trypanosoma cruzi, the agent of Chagas’ disease. NO is a key mediator of parasite killing in mice experimentally infected with T. cruzi, and previous studies have suggested that leukotrienes, such as LTB₄, induces NO synthesis in T. cruzi-infected macrophages and plays a relevant role in the killing of parasite in a NO-dependent manner. We therefore investigated whether leukotrienes would have a similar role in vivo in controlling the parasite burden by regulating NO activity. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased NO and IL-6 production in the plasma with a concomitant decrease in the expression of iNOS in the cardiac tissue on day 12 after T. cruzi infection. These findings indicate that endogenous leukotrienes are important regulators of NO activity in the heart and therefore influence the cardiac parasite burden without exerting a direct action on IL-6 production in the acute phase of infection with T. cruzi.     

Nitric oxide modulates the expression of endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO^•). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO^• to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration.Effects of NO^•, either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO^• down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO^• was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression.In conclusion, during angiogenesis and leukocyte recruitment, NO^• regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO^•, reflecting the functions of the tissue they originate from.     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.