Inhibition Of DNA Fragmentation Research Articles

Ephedra alata Seeds Confer Kidney Protection against Early Life Exposure to Acephate by Regulating Oxidative Insult and Activating Autophagy.

The aim of the current work was to examine for the first time the nephropreventive capacity of Ephedra alata seed extract (E) against maternal exposure to acephate in rat offspring. The in vivo results revealed that E. alata supplementation for 28 days (40 mg/kg b.w.) significantly attenuated the nephrotoxicity in adult offspring induced by acephate. In fact, it decreased the levels of creatinine and uric acid and increased the albumin content compared to the intoxicated group. The in utero studies showed that E. alata inhibited the renal oxidative stress generated by acephate exposure by reducing lipid peroxidation and enhancing antioxidant biomarker activities (GSH, CAT, and SOD). The inhibition of DNA fragmentation and the improvement of the ultrastructural changes highlighted the prophylactic effect of E. alata in renal tissue. Additionally, the immunofluorescence study showed the upregulation of LC3 gene expression, suggesting the capacity of E. alata extract to stimulate autophagic processes as a protective mechanism. Molecular docking analysis indicated that hexadecasphinganine, the major compound in E. alata, has a higher affinity toward the Na+/K+-ATPase, epithelial sodium channel (ENaC), and sodium hydrogen exchanger 3 (NHE3) genes than acephate. Hexadecasphinganine could be considered a potential inhibitor of the activity of these genes and therefore exerted its preventive capacity. The obtained findings confirmed that E. alata seed extract exerted nephropreventive capacities, which could be related to its bioactive compounds, which possess antioxidant activities.

Leonotis ocymifolia (Burm.f.) Iwarsson aerial parts aqueous extract mitigates cisplatin-induced nephrotoxicity via attenuation of inflammation, and DNA damage.

Herein, we explored the protective effect of Leonotis ocymifolia (Burm.f.) Iwarsson aerial parts extract (LO) against cisplatin (CP)-induced nephrotoxicity in rats and profiled their phytocontents. A total of 31 compounds belonging to organic and phenolic acids and their glycosides as well as flavonoids and their O- and C-glycosides were identified through LC-MS/MS. The DPPH and FRAP assays revealed that the extract had powerful antioxidant properties. The in vivo results demonstrated that administering LO extract for 30days (40 and 80mg/kg b. w.) significantly improved the altered renal injury markers via reducing creatinine (high dose only) and uric acid levels compared to the Cp-group. The deleterious action of cisplatin on renal oxidative stress markers (GSH, MDA, SOD, and CAT) were also mitigated by LO-pretreatment. The reduction of the inflammatory marker (IL-6), and inhibition of DNA fragmentation, highlighted the prophylactic action of LO in kidney tissue. Molecular docking followed by a 100 ns molecular dynamic simulation analyses revealed that, amongst the 31 identified compounds in LO, chlorogenic and caffeoylmalic acids had the most stable binding to IL-6. The nephroprotective effects were further confirmed by histopathological observations, which showed improvement in ultrastructural changes induced by cisplatin. The observed findings reinforce the conclusion that L. ocymifolia extract exerts nephroprotective properties, which could be related to its antioxidant and anti-inflammatory activities. Further studies are required to determine the therapeutic doses and the proper administration time.

Berberine chloride protects cochlear hair cells from aminoglycoside-induced ototoxicity by reducing the accumulation of mitochondrial reactive oxygen species

Aminoglycoside, a medicinal category of antibiotics, are used in treatment of Gram-negative bacterial infections. Although they are the most widely-used antibiotics due to their high efficacy and low cost, several main adverse effects have been reported including nephrotoxicity and ototoxicity. Since drug-induced ototoxicity is one of the major etiological causes of acquired hearing loss, we examined cochlear hair cell damages caused by three aminoglycosides (amikacin, kanamycin, and gentamicin), and investigated protective property of an isoquinoline-type alkaloid, Berberine chloride (BC). Berberine, a well-known bioactive compound found from medicinal plants, has been known to have anti-inflammatory, antimicrobial effects. To determine protective effect of BC in aminoglycoside-induced ototoxicity, hair cell damages in aminoglycoside- and/or BC-treated hair cells using ex vivo organotypic culture system of mouse cochlea. Mitochondrial ROS levels and depolarization of mitochondrial membrane potential were analyzed, and TUNEL assay and immunostaining of cleaved caspase-3 were performed to detect apoptosis signals. As the results, it was found that BC significantly prevented aminoglycoside-induced hair cell loss and stereocilia degeneration by inhibiting excessive accumulation of mitochondrial ROS and subsequent loss of mitochondrial membrane potential. It eventually inhibited DNA fragmentation and caspase-3 activation, which were significant for all three aminoglycosides. This study is the first report suggested the preventative effect of BC against aminoglycoside-induced ototoxicity. Our data also suggests a possibility that BC has the potential to exert a protective effect against ototoxicity caused by various ototoxic drugs leading to cellular oxidative stress, not limited to aminoglycoside antibiotics.

Beneficial Effects of Bauhinia rufa Leaves on Oxidative Stress, Prevention, and Treatment of Obesity in High-Fat Diet-Fed C57BL/6 Mice.

Obesity is an epidemic disease worldwide, associated with oxidative stress and the development of several other diseases. Bauhinia rufa (Bong.) Steud. is a native Brazilian Cerrado medicinal plant popularly used for the treatment of obesity. In this context, we investigated the chemical composition of the methanolic extract of B. rufa leaves (MEBr) and evaluated the antioxidant activity and its impact on the prevention and treatment of obesity in mice fed a high-fat diet (HFD 60%). Additionally, the acute oral toxicity of MEBr was evaluated. In MEBr, 17 glycosylated compounds were identified, including myricetin, quercetin, kaempferol, coumaroyl, cyanoglucoside, and megastigmane. In vitro, MEBr showed antioxidant activity in different methods: DPPH•, ABTS•+, FRAP, iron-reducing power, inhibition of β-carotene bleaching, and inhibition of DNA fragmentation. In human erythrocytes, MEBr increased the activities of antioxidant enzymes, superoxide dismutase, and catalase. Under oxidative stress, MEBr reduced oxidative hemolysis, and the malondialdehyde (MDA) levels generated in erythrocytes. Mice treated acutely with MEBr (2000 mg/kg) showed no signs of toxicity. During 90 days, the mice received water or MEBr simultaneously with HFD for induction of obesity. At this stage, MEBr was able to reduce the gain of subcutaneous white adipose tissue (WAT) and prevent the increase of MDA in the heart and brain. After 180 days of HFD for obesity induction, mice that received MEBr simultaneously with HFD (HFD-MEBr) in the last 60 days of treatment (120-180 days) showed a reduction of retroperitoneal and mesenteric WAT deposits and MDA levels in the heart, liver, kidney, and brain, compared to the HFD-Control group. These effects of MEBr were similar to mice treated with sibutramine (HFD-Sibutramine, 2 mg/kg). Combined, the results show that compounds from the leaves of B. rufa affect controlling oxidative stress and actions in the prevention and treatment of obesity. Thus, associated oxidative stress reduction and body composition modulation, in obese people, can contribute to the prevention of obesity-related comorbidities and improve quality of life.

Hepatoprotective activity of raspberry ketone is mediated via inhibition of the NF-κB/TNF-α/caspase axis and mitochondrial apoptosis in chemically induced acute liver injury.

Raspberry Ketone (RK) is a natural phenolic compound which is marketed nowadays as a popular weight-reducing remedy, with reported antioxidant and anti-inflammatory activities. However, its biological activity is not fully elucidated. Hepatotoxicity is the leading cause of acute liver failure in Europe and North America, and its management is still challenging. Therefore, this study aimed to assess the therapeutic detoxification activity of RK against liver injury in vivo and to explore the underlying mechanisms using carbon tetrachloride (CCl4)-induced hepatotoxicity as a model. First, a dose-response study using 4 different doses, 25, 50, 100, and 200 mg kg-1 day-1, of RK was conducted. RK was administered for 5 days as a pretreatment, followed by a single dose of CCl4 (1 ml kg-1, 1 : 1 v/v CCl4 : olive oil). The RK dose of 200 mg kg-1 showed the greatest protective effect and was selected for further investigations. CCl4 hepatotoxicity was confirmed by elevation of liver enzymes, and histopathological examination. CCl4-induced oxidative stress was evident from increased lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS) along with depleted superoxide dismutase (SOD), reduced glutathione (GSH), and total antioxidant capacity (TAC). Increased oxidative stress was associated with increased cytochrome c expression with subsequent activation of caspase-9 and caspase-3, in addition to DNA fragmentation reflecting apoptosis. CCl4 also induced the expression of inflammatory cytokines (NF-κB and TNF-α). Interestingly, RK hepatoprotective activity was evident from the reduction of liver enzymes, and maintenance of hepatocyte integrity and microstructures as evaluated by histopathological examination using H and E, and transmission electron microscopy. The antioxidant activity of RK was demonstrated by the increase of TAC, SOD, and GSH, with a concomitant decrease of the TBARS level. Moreover, RK pretreatment inhibited CCl4-induced upregulation of inflammatory mediators. RK antiapoptotic activity was indicated by the reduction of the expression of cytoplasmic cytochrome-C, a decrease of caspases, and inhibition of DNA fragmentation. In conclusion, this study demonstrates that RK is a promising hepatoprotective agent. The underlying mechanisms include antioxidant, anti-inflammatory, and anti-apoptotic activities. This is the first study reporting RK hepatoprotective activity in acute hepatic injury and approves its antiapoptotic effect in the liver.

HN1L is essential for cell growth and survival during nucleopolyhedrovirus infection in silkworm, Bombyx mori

Hematological and neurological expressed 1-like (HN1L) protein is an evolutionarily conserved protein that plays an important role in embryonic development. It has been reported that HN1L is involved in the process of cell growth and cancer formation and that cell cycle arrest occurs during suppression of HN1L expression. Previous studies have demonstrated that the expression levels of the Bombyx mori HN1L protein were significantly downregulated in Bombyx mori Nucleopolyhedrovirus (BmNPV) infected silkworm cells. Transient transfections were performed with plasmids for pIEX-1-HN1L expression in Bombyx mori ovarian cells (BmN) in order to explore the effect of the HN1L protein on the growth of silkworm cells and its regulatory role in the process of viral infection. Cellular localization analysis revealed that HN1L was localized in the cytoplasm and that its upregulation could significantly enhance cellular activity. Furthermore, HN1L could promote G1/S phase conversion, thereby contributing to cell proliferation. Upon infection of BmN cells with BmNPV, the induction of apoptosis increased, although HN1L overexpression could inhibit DNA fragmentation, suggesting that the HN1L protein could inhibit cell apoptosis induced by viral invasion. In addition, Western blotting indicated that the HN1L protein inhibited the activation of caspase-9 zymogen and the expression of Bax protein, although it promoted Bcl-2 expression. Flow cytometry analysis further confirmed that overexpression of HN1L significantly inhibited apoptosis induced by BmNPV infection. Consequently, we demonstrated that BmN HN1L is a protein with multiple functions, which enhanced cell activity, regulated the cell cycle and induced an anti-apoptotic response by BmNPV infection.

Mycoplasma bovis delay in apoptosis of macrophages is accompanied by increased expression of anti-apoptotic genes, reduced cytochrome C translocation and inhibition of DNA fragmentation

Bacterial pathogens have evolved to manipulate host cell death and survival pathways for their intracellular persistence. Understanding the ability of a bacterium to induce or inhibit cell death is essential for elucidating the disease pathogenesis and suggesting potential therapeutic options to manage the infection. In recent years, apoptosis inhibition by different bacteria has been suggested as a mechanism of survival by allowing the pathogen to replicate and disseminate in the host. Mycoplasma bovis has evolved mechanisms to invade and modulate apoptosis of bovine peripheral blood mononuclear cells (PBMC), red blood cells (RBCs), primary macrophages and monocytes. To date, these mechanisms are poorly understood. Using apoptosis assays such as Annexin V binding, caspases activity, reactive oxygen species production, DNA fragmentation and differential gene expression we set out to determine how M. bovis modulates macrophage survival. Using the BoMac cell line, we report a significant reduction in STS-induced apoptosis through caspase dependent manner. Besides activating the NF-kβ pathway and inhibiting caspases 3, 6 and 9, M. bovis strain Mb1 also inhibits production of reactive oxygen species and DNA fragmentation of the host cell. We also report a significant up-regulation of the anti-apoptotic genes Bcl-2 and Bcl-XL upon infection. Our results indicate that M. bovis strain Mb1 inhibits the intrinsic pathway of apoptosis and up-regulate survival genes in BoMac cells.

Role of Leptin/Osteopontin Axis in the Function of Eosinophils in Allergic Rhinitis with Obesity.

Background Allergic rhinitis (AR) is characterized by tissue and blood eosinophilia. Previous studies showed enhanced eosinophilia in allergic rhinitis patients with obesity, suggesting an association between obesity and eosinophilia. However, the interaction and mechanism between obesity and eosinophilia is still unclear. Methods We recruited thirty AR children and 30 controls in this study. Expression of leptin and osteopontin (OPN) proteins in serum was detected, and correlation analysis with eosinophilia was performed. The effect of leptin or OPN on eosinophil apoptosis, adhesion, migration, and activation of eosinophil was examined. Ovalbumin-sensitized mice were established to prove the role of obesity on eosinophil regulation by leptin and OPN. Results We found that upregulated serum and nasal leptin and OPN expression in AR were positively correlated with eosinophilia and eosinophil cationic protein levels. Leptin or OPN inhibited eosinophil apoptosis, demonstrated as inhibited DNA fragmentation and phosphatidylserine (PS) redistribution (P < 0.05). Leptin and OPN promote expression of cluster of differentiation 18 (CD-18) and intercellular adhesion molecule 1 (ICAM-1) and inhibit expression of ICAM-1 and L-selectin by eosinophils, which contribute to the adhesion of eosinophils. Leptin and OPN mediated migration and activation of eosinophil through phosphatidylinositol-3-OH kinase (PI3K) pathway. Obese AR mice presented with more severe eosinophilia and symptoms compared with nonobese AR mice or control mice. Immunochemistry staining of leptin and OPN of nasal turbinate in obese AR mice was also stronger than those in nonobese AR mice or control mice. Anti-OPN, anti-leptin, and anti-α4 treatments reduce nasal eosinophilia inflammation and clinical symptoms in model mice. Conclusion Our results suggested that in an obese state, upregulation of leptin and OPN regulates apoptosis, adhesion, migration, and activation of eosinophils, and this process may be mediated by the PI3K and anti-α4 pathways.

The molecular and biochemical insight view of lycopene in ameliorating tramadol-induced liver toxicity in a rat model: implication of oxidative stress, apoptosis, and MAPK signaling pathways.

The influence of tramadol (TD) on hepatic tissue and the potential efficiency of lycopene to mitigate TD-induced hepatotoxic impacts were determined. Forty male albino rats were allocated into four groups: group I, untreated (placebo); group II, injected with TD (15mgkg-1) intraperitoneally (i.p.); group III, gastrogavaged with lycopene (10mgkg-1) per os (p.o.); and group IV received TD with lycopene with the same mentioned doses for 15days. The results demonstrated that TD induced augmentation in tissue lipid peroxidation biomarker and disturbance in the antioxidant homeostasis and elevated the activity of serum liver injury biomarkers and decreased serum protein, globulin, and albumin. Hepatic glutathione S-transferase (GST), superoxide dismutase (SOD), thioredoxin-1 (Txn-1), and catalase (CAT) activities and gene expression were decreased and glutathione content was reduced in the TD-challenged rats, and these effects were alleviated by lycopene. Furthermore, TD induced apoptosis in liver tissues as shown by DNA fragmentation and upregulation of proapoptotic Bax and Casp-3 while lycopene upregulated the antiapoptotic Bcl-2. The results of Western blot showed that lycopene initiated low expression of mitogen activated protein kinase pathway (MAPK) protein expression in liver tissues of TD-challenged rats. In addition, lycopene reduced fatty degeneration and necrosis of the liver in TD-challenged group. Our data demonstrate that lycopene appears to be highly efficient in mitigating the hepatotoxic impacts of TD by preventing lipid peroxidation and initiating modifications in the expression and activity of antioxidant pathways. Surprisingly, lycopene fortified liver tissue by inhibiting DNA fragmentation and apoptosis signaling induced by TD. MAPK activation may be dependent from ROS generation; due to lycopene which possessed antioxidant potential did have a substantial effect on MAPK activity.

FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes

BackgroundFAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in insulin-secreting beta-cells. Since in silico data revealed that FAM3B can be expressed in prostate tumors, we evaluated the putative role of this cytokine in prostate tumor progression.MethodsFAM3B expression was analyzed by quantitative PCR in tumor tissue clinical samples and prostate tumor cell lines. Culture growth and viability of DU145 cell line were evaluated after treatment with either exogenous FAM3B protein obtained from conditioned media (CM) of 293 T cells overexpressing FAM3B or a recombinant FAM3B protein produced in a bacterial host. DU145 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145/FAM3B cells after treatment with several cell death inducers, such as TNF-alpha, staurosporine, etoposide, camptothecin, and serum starvation conditions. Anchorage-independent growth in soft agarose assay was used to evaluate in vitro tumorigenicity. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice.ResultsWe observed an increase in FAM3B expression in prostate tumor samples when compared to normal tissues. DU145 cell viability and survival increased after exogenous treatment with recombinant FAM3B protein or FAM3B-secreted protein. Overexpression of FAM3B in DU145 cells promoted inhibition of DNA fragmentation and phosphatidylserine externalization in a time and dose-dependent fashion, upon apoptosis triggered by TNF-alpha. These events were accompanied by increased gene expression of anti-apoptotic Bcl-2 and Bcl-XL, decreased expression of pro-apoptotic Bax and diminished caspase-3, −8 and −9 proteolytic activities. Furthermore, inhibition of Bcl-2 anti-apoptotic family proteins with small molecules antagonists decreases protective effects of FAM3B in DU145 cells. When compared to the respective controls, cells overexpressing FAM3B displayed a decreased anchorage- independent growth in vitro and increased tumor growth in xenografted nude mice. The immunohistochemistry analysis of tumor xenografts revealed a similar anti-apoptotic phenotype displayed by FAM3B-overexpressing tumor cells.ConclusionsTaken together, by activating pro-survival mechanisms FAM3B overexpression contributes to increased resistance to cell death and tumor growth in nude mice, highlighting a putative role for this cytokine in prostate cancer progression.

Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis.

Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we studied whether Nrf2 exerts chondroprotective effects by suppressing the oxidative stress and apoptosis in IL-1β stimulated human OA chondrocytes. Expression of Nrf2 and its target genes HO-1, NQO1 and SOD2 was significantly high in OA cartilage compared to normal cartilage and was also higher in damaged area compared to smooth area of OA cartilage of the same patient. Human chondrocytes treated with IL-1β resulted in robust Nrf2/ARE reporter activity, which was inhibited by pretreatment with antioxidants indicating that Nrf2 activity was due to IL-1β-induced ROS generation. Ectopic expression of Nrf2 significantly suppressed the IL-1β-induced generation of ROS while Nrf2 knockdown significantly increased the basal as well as IL-1β-induced ROS levels in OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1β-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction and mitochondrial ROS production in OA chondrocytes. Nrf2 over-expression in OA chondrocytes increased the expression of anti-apoptotic proteins while pro-apoptotic proteins were suppressed. Importantly, Nrf2 over-expression activated ERK1/2 and its downstream targets-ELK1, P70S6K and P90RSK and suppressed the IL-1β-induced apoptosis whereas inhibition of ERK1/2 activation abrogated the protective effects of Nrf2 in OA chondrocytes. Taken together, our data demonstrate that Nrf2 is a stress response protein in OA chondrocytes with anti-oxidative and anti-apoptotic function and acts via activation of ERK1/2/ELK1-P70S6K-P90RSK signaling axis. These activities of Nrf2 make it a promising candidate for the development of novel therapies for the management of OA.

Isolation and screening of rare Actinobacteria, a new insight for finding natural products with antivascular calcification activity.

Vascular calcification (VC) is a significant pathological process in some life-threatening diseases. Several pathological mechanisms, including transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and apoptosis are involved in VC. Compounds with an inhibitory effect on these processes are potentially efficient medications. In consideration of the multiple biological activities of Actinobacteria, this research was aimed at finding anti-VC metabolite-producing Actinobacteria. After the isolation and identification of Actinobacteria, the effect of their fermentation broth extracts on the apoptosis rate was measured using various methods, for example, ethidium bromide/acridine orange staining, DNA laddering and diphenylamine assays. The effect of the most effective fermentation broth extract of Actinobacteria (FBEA) on the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) was examined. Finally, the most effective FBEA was fractionated and the chemical composition of anti-VC fractions was analysed using GC-MS. Various VC inhibition rates were observed in the tested FBEA (20μgml-1 ; 17·9-60·15%). The inhibition of DNA fragmentation was 7-48%. The FBE with the greatest anticalcification activity belonged to Kribbella sp. UTMC 267 and, according to 16S rRNA analysis, Kribbella sancticallisti with a similarity of 98·53% is its nearest neighbour. The FBE of Kribbella sp. UTMC 267 reduced Runx2 mRNA expression by 2·95-fold and OPN mRNA expression by 28·57-fold, both of which are considered significant (P<0·05). Finally, GC-MS analysis showed the existence of potent anti-oxidative and anti-inflammation agents in FBE of Kribbella sp. UTMC 267. Actinobacterial metabolites can provide a new strategy for treating VC diseases by reducing the expression of osteogenic genes, the apoptosis rate and oxidative stress. This study highlights the therapeutic potential of Kribbella sp. metabolites and Actinobacteria as a new natural source for drug discovery programs in the nonantibiotic bioactivity field.

Dacryodes edulis enhances antioxidant activities, suppresses DNA fragmentation in oxidative pancreatic and hepatic injuries; and inhibits carbohydrate digestive enzymes linked to type 2 diabetes

The leaves of Dacryodes edulis were investigated for their anti-oxidative and anti-diabetic potentials in vitro. Extracts from sequential extraction with solvents of increasing polarity (n-hexane, ethyl acetate, ethanol and aqueous) of the leaves were subjected to in vitro antioxidant assays using the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging and Ferric reducing antioxidant power (FRAP) protocols respectively. Their inhibitory effects were investigated on α-glucosidase, pancreatic lipases, pancreatic ATPase and glucose-6-phospatase activities. Their antioxidant and anti-apoptotic effects on Fe2+ − induced oxidative injuries in pancreatic and hepatic tissues were also investigated ex vivo. The ethanol extract was subjected to Gas chromatography mass spectroscopy (GC–MS) and Fourier transform infrared (FTIR) spectroscopic analysis to identify its bioactive chemical constituents. The extracts showed potent free radical scavenging activity and significantly (p<0.05) inhibited all studied enzymes, with the ethanol extract showing greater activities. Superoxide Dismutase (SOD) and Catalase (CAT) activities were significantly (p<0.05) increased in both pancreatic and hepatic tissues with concomitant elevation of reduced glutathione (GSH) levels as well as reduced levels of malondialdehyde (MDA). The extracts significantly inhibited DNA fragmentation. These activities were dose − dependent. Amongst compounds identified, only Kaur-15-ene, Urs-12-ene-3-ol acetate and 2,3,23-trihydroxyolean-12-en-28-oic acid methyl ester showed strong binding affinities when docked with α-glucosidase (PDB ID:3TON). These results indicate the anti-oxidative, anti-diabetic and anti-obesogenic potentials of D. edulis leaves, which gives credence to its antidiabetic folkloric claims.

Pleiotropic Roles of Nrf2 as Regulators of Chondrocyte Apoptosis, Oxidative Stress, Inflammatory Response and Catabolic and Anabolic Pathways in Osteoarthritis

Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we investigated the regulation of oxidative stress, inflammatory response, apoptosis, catabolic and anabolic factors by Nrf2 in IL-1β-stimulated human OA chondrocytes. Ectopic expression of Nrf2 in OA chondrocytes suppressed the generation of ROS while Nrf2 knockdown significantly increased the ROS levels in IL-1β-stimulated OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1β-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction, suppression of pro-apoptotic proteins Bax, Bad and Bid expression and enhanced expression of anti-apoptotic proteins Bcl2, Bcl-xl and Mcl-1 in OA chondrocytes. To examine the role of Nrf2 in IL-1β-induced inflammatory response, we determined the mRNA expression and protein levels of several cytokines and chemokines using PCR array and multiplex immunoassay. Our data demonstrate that Nrf2 activation inhibited the IL-1β-mediated expression of several inflammatory mediators and Gene Ontology analysis revealed significant enrichment of MAPK signaling, TNF signaling, NF-κβ signaling and cytokine receptor signaling pathways. Additionally, Nrf2 activation inhibited the expression matrix degrading proteases-MMP-13, 3, 9, and ADAMTS-4 and enhanced the expression of OA related anabolic factors-COL2A1, ACAN, FRZB and GDF5 and SOX-5, 8 and 9 in IL-1β-stimulated OA chondrocytes. Importantly, we also discovered that Nrf2 mediated suppression of catabolic factors was dependent on ERK1/2 activation by Nrf2. Our data demonstrate that Nrf2 regulates multiple pathways involved in chondrocyte apoptosis, inflammation and matrix degradation via activation of ERK1/2/ELK1-P70S6K-P90RSK axis. Our findings collectively support the development of therapies targeting Nrf2 for the management of OA.

MiR-146a suppresses STAT3/VEGF pathways and reduces apoptosis through IL-6 signaling in primary human retinal microvascular endothelial cells in high glucose conditions

microRNA (miRNA) play critical roles in the pathological processes of diabetic retinopathy, including inflammatory responses, insulin signaling, and angiogenesis.In addition to their regulatory functions on gene expression, miRNA is considered as a potential therapeutic target, as well as a diagnostic marker for many diseases. Our understanding on the pathological mechanisms underlying diabetic retinopathy is still incomplete and additional investigations are required to develop novel therapeutic strategies. The aim of this study was to investigate our hypothesis that miR-146a plays a role in suppressing pro-inflammatory pathways, involving STAT3 and VEGF, through regulating IL-6 signaling to reduce apoptosis of human retinal endothelial cells (REC) in high glucose conditions. Human REC were cultured in normal (5mM) glucose or high glucose medium (25mM) for 3days. We performed transfections on REC with miRNA mimics (hsa-miR-146a-5p). Overexpression of miR-146a reduced IL-6 levels, STAT3 phosphorylation, and VEGF levels in REC cultured in high glucose. Cellular apoptosis was decreased in REC overexpressing miR-146a, as demonstrated by the inhibition of DNA fragmentation. More importantly, we demonstrated that the regulatory role of miR-146a on STAT3/VEGF and apoptosis was mediated by IL-6 receptor signaling in REC.Overall, we report that miR-146a suppressed IL-6 signaling, leading to reduced levels of STAT3 and VEGF in REC in high glucose conditions, leading to decreased apoptosis. The outcome suggests that miR-146a is a potential molecular target for inhibiting inflammation and apoptosis in the diabetic retina through the suppression of the IL-6-mediated STAT3/VEGF pathway.

Protective effect of Tritone (Livosone) on oxidative DNA damage and its hepatoprotective potential against various hepatotoxic agent in wistar rats

AimTo evaluate antioxidant activity, DNA damage inhibition and hepatoprotecitve potential of polyherbal formulation Tritone (Livosone). MethodsIn vitro antioxidant activity of Tritone formulation was performed by using DPPH assay. Hepatoprotecitve potential of Tritone was evaluated against various hepatotoxic agents including Paracetamol (2g/kg b. wt p.o. single dose on 15th day), Galactosamine (400mg/kg b. wt. i.p. single dose on 8th day) and Alcohol (30% p.o.1ml/100g of rat for 15days). Tritone formulation at the doses of (40.5, 81 and 162mg/kg) and standard silymarin (100mg/kg) and Liv52 (270mg/kg) were administered p.o. The hepatoprotective assessment was done by estimating biochemical parameters: SGOT, SGPT, ALP and Total Bilirubin total protein and ChE levels. Additionally histopathological and DNA fragmentation study of Tritone was also performed. ResultAdministration of hepatotoxins (paracetamol, D-GaiN and alcohol) in experimental animals showed significant biochemical, histological deterioration and DNA fragmentation. Pretreatment with Tritone (Livosone) shows significant reduction in serum SGOT, SGPT, ALP and total bilirubin levels and shows significant elevation in total protein and cholinesterase (ChE) levels compared to groups treated with hepatotoxic agents. Histopathological observations of rat liver pretreated with Tritone (Livosone) shows significant protection against hepatic damage. Inhibition of DNA fragmentation by Tritone indicates protective effect of formulation on liver at molecular level. Finally all the results were compared with standard drugs Silymarin and Liv52. ConclusionCorrelation of antioxidant activity, biochemical results, histopathological changes and inhibition of DNA damage after treatment with Tritone shows maximum hepatoprotective potential at dose 81mg/kg and 162mg/kg.

Epigallocatechin-3-gallate prevents cardiac apoptosis by modulating the intrinsic apoptotic pathway in isoproterenol-induced myocardial infarction

(−)Epigallocatechin-gallate (EGCG) is an emerging natural therapy. This study examined the cardioprotective effect of EGCG on isoproterenol-induced myocardial damage and apoptosis and EGCG's role in modulating the expression of apoptotic signaling proteins. Experimental myocardial infarction was induced in albino Westar rats by isoproterenol (ISO) administration (100mg/kg, s.c.) at an interval of 24h on the 6th and 7th day. EGCG (15mg/kg, i.p.) was administered seven days before ISO. EGCG pretreatment significantly showed an anti-lipidemic effect and protected the cell membrane integrity, as shown by the blocking of changes in serum levels of CK-MB, LDH, ALP, ALT and troponin T. EGCG also maintained the redox balance by preventing the inhibition of the activity of SOD and CAT while limiting lipid peroxidation. Pretreatment with EGCG inhibited the stimulation of the pro-inflammatory cytokine, TNF-α, in the serum. In animals treated with EGCG, tissue Bcl-2 expression exceeded the values observed after ISO treatment and down-regulated the expression of pro-apoptotic signaling proteins, including Bax, caspase-9 and 3. This is accompanied by the protection of genomic integrity by inhibiting DNA fragmentation coincident with the down-regulation of P53. In conclusion, EGCG protected against cardiac damage by decreasing apoptosis in myocardium tissue by 1) maintaining the balance of anti-apoptotic / pro-apoptotic signaling proteins in the mitochondrial pathway of cell death, 2) limiting oxidative stress while performing antioxidant and anti-inflammatory effects, and 3) protecting DNA integrity, sustaining cardiac health. Therefore, EGCG is potentially beneficial as an early intervention in cardiac attack.

LPA1 Mediates Antidepressant-Induced ERK1/2 Signaling and Protection from Oxidative Stress in Glial Cells.

Antidepressants have been shown to affect glial cell functions and intracellular signaling through mechanisms that are still not completely understood. In the present study, we provide evidence that in glial cells the lysophosphatidic acid (LPA) receptor LPA1 mediates antidepressant-induced growth factor receptor transactivation, ERK1/2 signaling, and protection from oxidative stress. Thus, in C6 glioma cells and rat cortical astrocytes, ERK1/2 activation induced by either amitriptyline or mianserin was antagonized by Ki16425 and VPC 12249 (S), which block LPA1 and LPA3 receptors, and by AM966, which selectively blocks LPA1 Cell depletion of LPA1 with siRNA treatment markedly reduced antidepressant- and LPA-induced ERK1/2 phosphorylation. LPA1 blockade prevented antidepressant-induced phosphorylation of the transcription factors CREB and Elk-1. Antidepressants and LPA signaling to ERK1/2 was abrogated by cell treatment with pertussis toxin and by the inhibition of fibroblast growth factor (FGF) receptor (FGF-R) and platelet-derived growth factor receptor (PDGF-R) tyrosine kinases. Both Ki16425 and AM966 suppressed antidepressant-induced phosphorylation of FGF-R. Moreover, blockade of LPA1 or inhibition of FGF-R and PDGF-R activities prevented antidepressant-stimulated Akt and GSK-3β phosphorylations. Mianserin protected C6 glioma cells and astrocytes from apoptotic cell death induced by H2O2, as indicated by increased cell viability, decreased expression of cleaved caspase 3, reduced cleavage of poly-ADP ribose polymerase and inhibition of DNA fragmentation. The protective effects of mianserin were antagonized by AM966. These data indicate that LPA1 constitutes a novel molecular target of the regulatory actions of tricyclic and tetracyclic antidepressants in glial cells.

Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma - Tissue Microarray of Human Samples.

Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of “inhibitor of caspase-activated DNase” (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

Hepatoprotective Effects and Mechanisms of Action of Triterpenoids from Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) on α-Amanitin-Induced Liver Injury in Mice.

Most fatal mushroom poisonings are caused by species of the genus Amanita; the amatoxins are responsible for acute liver failure and death in humans. Ganoderma lucidum is a well-known traditional medicinal mushroom that has been shown to have obvious hepatoprotective effects. This study evaluated the hepatoprotective effects of triterpenoids from G. lucidum on liver injury induced by a-amanitin (α-AMA) in mice and the mechanisms of action of these triterpenoids, including radical scavenging and antiapoptosis activities. Mice were treated with α-AMA, followed by G. lucidum total triterpenoids or individual triterpenoids, and their hepatoprotective effects were compared with those of the reference drug silibinin (SIL). Treatment with SIL, G. lucidum total triterpenoids, and each of the 5 individual triterpenoids significantly reduced serum alanine aminotransaminase and aspartate ami- notransaminase concentrations and reduced mortality rates 20-40%. Moreover, triterpenoids and SIL significantly enhanced superoxide dismutase and catalase activity and reduced malondialdehyde content in livers. Treatment with ganoderic acid C2 significantly inhibited DNA fragmentation and decreased caspase-3, -8, and -9 activities. The results demonstrated that triterpenoids have hepatoprotective effects on α-AMA-induced liver injury and that their hepatoprotective mechanisms may be the result of their antioxidative and radical scavenging activities and their inhibition of apoptosis.