GSK-3ß/Notch-1 Activation Promotes Radiation-Induced Renal Damage: The Role of Gallic Acid in Mitigation of Nephrotoxicity.

Despite the therapeutic advances in treating malignancies, the efficient radiotherapeutic approaches with deprived adverse reactions still represent a potential clinical inquiry. The current study aims to elucidate the role of gallic acid (GA) in modifying the hazardous renal cytotoxicity induced by acute exposure to radiation. The MTT test was used to evaluate the viability of Vero cells exposed to 2 Gy gamma radiation with or without incubation of GA. In an in vivo model, male Wistar rats were divided into four experimental groups (n = 6): Control, Irradiated (IRR, 5 Gy), GA (100 mg/kg, i.p.) + IRR, and Glycogen synthase kinase inhibitor (GSKI, 3 mg/kg, i.p.) + IRR. Based on the MTT toxicity assay, from 0 and up to 5 µM dosages of GA did not demonstrate any cytotoxicity to Vero cells. The optimal GA dose that could protect the cells from radiation was 5 µM. Furthermore, GA exerted a protective effect from gamma radiation on renal tissue as indicated by corrected renal functions, decreased LDH level in serum, and balanced oxidative status, which is indicated by decreased tissue contents of NOx and TBARS with a significant increase of reduced GSH. These outcomes were inferred by the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) expression. The overall molecular impact of radiation in damaging the renal tissue may be explained by modifying the upstream AKT activity and its downstream targets GSK-3ß/Notch-1. Here, we concluded that the anticipated adverse reaction in the course of radiation exposure could be protected by daily administration of GA.

Pelargonidin ameliorates reserpine-induced neuronal mitochondrial dysfunction and apoptotic cascade: a comparative in vivo study.

BACKGROUND: Targeting the neuronal mitochondria as a possible intervention to guard against neurodegenerative disorder progression has been investigated in the current work via the administration of pelargonidin (PEL) to rats intoxicated by the mitochondrial toxin reserpine. The main criteria for choosing PEL were its reported antioxidant, anti-apoptotic and anti-inflammatory activities. METHODS: Male albino Wistar rats were randomized into five experimental groups; normal control, reserpinized to induce mitochondrial failure, standard PARP-1-inhibitor 1,5-isoquinolinediol (DIQ)-treated reserpinized, PEL-treated reserpinized, and GSK-3ß inhibitor (AR-A 014418) -treated reserpinized. RESULTS: PEL administration reversed the reserpine-induced abnormal behaviors marked by decreased catalepsy time. In addition, PEL restored brain glutathione with a reduction in nitric oxide content as compared to the reserpine-challenged group. Meanwhile, it improved neuronal mitochondrial function by the elevation of complex I activity associated with a low ADP/ATP ratio. Likely through its anti-inflammatory effect, PEL reduced the elevation of serum interleukin-1ß level and inhibited serum lactate dehydrogenase activity. These findings are aligned with the reduced expression of cleaved PARP and cleaved caspase-3 proteins, indicating PEL's suppressive effect on the intrinsic apoptotic pathway. Those biochemical findings were confirmed through comparable histopathological tissue examination among the experimental groups. CONCLUSIONS: In conclusion, PEL is a promising candidate for future use in the management of mitochondria-associated neuronal complications via controlling the ongoing inflammatory and degeneration cascades.

Poly(ADP-Ribose) Polymerase 1 Promotes Inflammation and Fibrosis in a Mouse Model of Chronic Pancreatitis.

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease's progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson's trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFß, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.

3-aminobenzamide, a poly (ADP ribose) polymerase inhibitor, enhances wound healing in whole body gamma irradiated model.

The custom use of radiotherapy was found to participate in the development of chronic unhealed wounds. In general, exposure to gamma radiation stimulates the production of reactive oxygen species (ROS) that eventually leads to damaging effect. Conversely, overexpression of a nuclear poly (ADP-ribose) polymerase enzyme (PARP) after oxidative insult extremely brings about cellular injury due to excessive consumption of NAD and ATP. Here, we dedicated our study to investigate the role of 3-aminobenzamide (3-AB), a PARP inhibitor, on pregamma irradiated wounds. Two full-thickness (6 mm diameter) wounds were created on the dorsum of Swiss albino mouse. The progression of wound contraction was monitored by capturing daily photo images. Exposure to gamma radiation (6Gy) exacerbated the normal healing of excisional wounds. Remarkably, topical application of 3-AB cream (50 µM) revealed a marked acceleration in the rate of wound contraction. Likewise, PARP inhibition ameliorated the unbalanced oxidative/nitrosative status of granulated skin tissues. Such effect was significantly revealed by the correction of the reduced antioxidant capacity and the enhanced lipid peroxidation, hydrogen peroxide, and myeloperoxidase contents. Moreover, application of 3-AB modified the cutaneous nitrite content throughout healing process. Conversely, the expressions of pro-inflammatory cytokines were down-regulated by PARP inhibition. The mitochondrial ATP content showed a lower consumption rate on 3-AB-treated wound bed as well. In parallel, the mRNA expressions of Sirt-1 and acyl-COA oxidase-2 (ACOX-2) were up-regulated; whom functions control the mitochondrial ATP synthesis and lipid metabolism. The current data suggested that inhibition of PARP-1 enzyme may accelerate the delayed wound healing in whole body gamma irradiated mice by early modifying the oxidative stress as well as the inflammatory response.

Activation of poly(ADP-ribose) polymerase-1 delays wound healing by regulating keratinocyte migration and production of inflammatory mediators.

Poly(ADP-ribosyl)ation (PARylation) is a protein modification reaction regulating various diverse cellular functions ranging from metabolism, DNA repair and transcription to cell death. We set out to investigate the role of PARylation in wound healing, a highly complex process involving various cellular and humoral factors. We found that topically applied poly[ADP-ribose] polymerase (PARP) inhibitors 3-aminobenzamide and PJ-34 accelerated wound closure in a mouse model of excision wounding. Moreover, wounds also closed faster in PARP-1 knockout mice as compared with wild-type littermates. Immunofluorescent staining for poly(ADP-ribose) (PAR) indicated increased PAR synthesis in scattered cells of the wound bed. Expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase and matrix metalloproteinase-9 was lower in the wounds of PARP-1 knockout mice as compared with control, and expression of IL-1ß, cyclooxygenase-2, TIMP-1 and -2 also were affected. The level of nitrotyrosine (a marker of nitrating stress) was lower in the wounds of PARP-1 knockout animals as compared with controls. In vitro scratch assays revealed significantly faster migration of keratinocytes treated with 3-aminobenzamide or PJ34 as compared with control cells. These data suggest that PARylation by PARP-1 slows down the wound healing process by increasing the production of inflammatory mediators and nitrating stress and by slowing the migration of keratinocytes.

Tricetin Reduces Inflammation and Acinar Cell Injury in Cerulein-Induced Acute Pancreatitis: The Role of Oxidative Stress-Induced DNA Damage Signaling.

Acute pancreatitis (AP) poses a worldwide challenge due to the growing incidence and its potentially life-threatening course and complications. Specific targeted therapies are not available, prompting the identification of new pathways and novel therapeutic approaches. Flavonoids comprise several groups of biologically active compounds with wide-ranging effects. The flavone compound, tricetin (TCT), has not yet been investigated in detail but sporadic reports indicate diverse biological activities. In the current study, we evaluated the potential protective effects of TCT in AP. TCT (30 µM) protected isolated primary murine acinar cells from the cytotoxic effects of cerulein, a cholecystokinin analog peptide. The protective effects of TCT were observed in a general viability assay (calcein ester hydrolysis), in an apoptosis assay (caspase activity), and in necrosis assays (propidium iodide uptake and lactate dehydrogenase release). The effects of TCT were not related to its potential antioxidant effects, as TCT did not protect against H2O2-induced acinar cell death despite possessing radical scavenging activity. Cerulein-induced expression of IL1ß, IL6, and matrix metalloproteinase 2 and activation of nuclear factor-κB (NFκB) were reduced by 30 µM TCT. In vivo experiments confirmed the protective effect of TCT in a mouse model of cerulein-induced AP. TCT suppressed edema formation and apoptosis in the pancreas and reduced lipase and amylase levels in the serum. Moreover, TCT inhibited interleukin-1ß (IL1ß), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) expression in the pancreas and reduced the activation of the oxidative DNA damage sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Our data indicate that TCT can be a potential treatment option for AP.

OGG1 Inhibition Reduces Acinar Cell Injury in a Mouse Model of Acute Pancreatitis.

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease with a complex pathology including oxidative stress. Oxidative stress triggers oxidative DNA lesions such as formation of 7,8-dihydro-8-oxo-2'-oxoguanine (8-oxoG) and also causes DNA strand breaks. DNA breaks can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) which contributes to AP pathology. 8-oxoG is recognized by 8-oxoG glycosylase 1 (OGG1) resulting in the removal of 8-oxoG from DNA as an initial step of base excision repair. Since OGG1 also possesses a DNA nicking activity, OGG1 activation may also trigger PARP1 activation. In the present study we investigated the role played by OGG1 in AP. We found that the OGG1 inhibitor compound TH5487 reduced edema formation, inflammatory cell migration and necrosis in a cerulein-induced AP model in mice. Moreover, TH5487 caused 8-oxoG accumulation and reduced tissue poly(ADP-ribose) levels. Consistent with the indirect PARP inhibitory effect, TH5487 shifted necrotic cell death (LDH release and Sytox green uptake) towards apoptosis (caspase activity) in isolated pancreatic acinar cells. In the in vivo AP model, TH5487 treatment suppressed the expression of various cytokine and chemokine mRNAs such as those of TNF, IL-1ß, IL1ra, IL6, IL16, IL23, CSF, CCL2, CCL4, CCL12, IL10 and TREM as measured with a cytokine array and verified by RT-qPCR. As a potential mechanism underlying the transcriptional inhibitory effect of the OGG1 inhibitor we showed that while 8-oxoG accumulation in the DNA facilitates NF-κB binding to its consensus sequence, when OGG1 is inhibited, target site occupancy of NF-κB is impaired. In summary, OGG1 inhibition provides protection from tissue injury in AP and these effects are likely due to interference with the PARP1 and NF-κB activation pathways.

Targeting Nuclear NAD+ Synthesis Inhibits DNA Repair, Impairs Metabolic Adaptation and Increases Chemosensitivity of U-2OS Osteosarcoma Cells.

Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1-/- cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of NAD+ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage (phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1-/- cells to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important for chemosensitization. Cisplatin-induced cell death of NMNAT1-/- cells was also characterized by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity, highlighting the central role of compromised cellular bioenergetics in chemosensitization by NMNAT1 inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is a promising therapeutic target for osteosarcoma and possibly other tumors as well.

Potential effects of ursodeoxycholic acid on accelerating cutaneous wound healing.

Among the initial responses to skin injury, triggering inflammatory mediators and modifying oxidative status provide the necessary temple for the subsequent output of a new functional barrier, fibroplasia and collagen deposition, modulated by NF-κB and TGF-ß1 expressions. Hence, the current study aimed to investigate the effect of local application of ursodeoxycholic acid (UDCA) on cutaneous wound healing induced in Swiss mice. Wound contraction progression was monitored by daily photographing the wounds. Enhanced fibroblast cell migration was observed after incubation with UDCA. Topical application of UDCA (500 µM) cream on excised wounds significantly enhanced wound contraction and improved morphometric scores. In addition, UDCA ameliorated the unbalanced oxidative status of granulated skin tissues. Interestingly, it showed increased expression of TGF-ß1 and MMP-2 with decreased expression of NF-κB. On the other hand, UDCA significantly increased collagen fibers deposition and hydroxyproline content and enhanced re-epithelization. UDCA also modified the mitochondrial function throughout the healing process, marked by lower consumption rates of mitochondrial ATP, complex I contents as well as intracellular NAD+ contents accompanied by elevated levels of nicotinamide compared to the untreated controls. In chronic gamma-irradiated (6Gy) model, the illustrated data showed enhanced wound contraction via increased TGF-ß1/MMP-2 and collagen deposition incurred by topical application of UDCA without effect on NF-κB level. In sum, the present findings suggest that UDCA may accelerate wound healing by regulating TGF-ß1 and MMP-2 and fibroplasia/collagen deposition in either the two wound healing models.

L-thyroxine modifies nephrotoxicity by regulating the apoptotic pathway: The possible role of CD38/ADP-ribosyl cyclase-mediated calcium mobilization.

Thyroid hormones are well-established as a key regulator of many cellular metabolic pathways developed in various pathogeneses. Here, we dedicated the current work to investigate the role of thyroid hormone analogue (L-thyroxine, L-TH) in regulating the renal cytotoxicity using in vivo and in vitro models. Swiss mice were exposed to gamma radiation (IRR, 6Gy) or treated with cisplatin (CIS, 15 mg/kg, i.p.) for induction of nephrotoxicity. Remarkably, pretreatment with L-TH (1µg/kg) ameliorated the elevated kidney function biomarkers, oxidative stress and protected the renal tissue from the subsequent cellular damage. Likewise, L-TH inhibited the apoptotic cascade by down-regulating the extreme consumption of the cellular energy (ATP), the expression of caspase-3 and Bax, and the stimulation of cyclic ADP ribose (cADPR)/calcium mobilization. Moreover, incubation with L-TH (120nM/4h) significantly blocked the cytotoxicity of CIS on Vero cells and the depletion of NAD+ content as well as modified the ADP-ribose cyclase (CD38) enzymatic activity. High doses of L-TH (up to30 nM/4h) inversely increased the radiosensitivity of Vero cells towards IRR (up to 6Gy). On the other hand, L-TH did not interfere CIS-induced cytotoxicity of colorectal adenocarcinoma (Caco-2) cell line. In conclusion, pretreatment with L-TH could be a promising protective approach to the renal cellular damage induced during either CIS or IRR therapy by regulating the unbalanced oxidative status, the expression of pro-apoptotic biomarkers via modulation of cADPR mediated-calcium mobilization.