Tissue-protective Activities Research Articles

Multifaceted Tissue-Protective Functions of Polyvalent Immunoglobulin Preparations in Severe Infections-Interactions with Neutrophils, Complement, and Coagulation Pathways.

Severe infections induce immune defense mechanisms and initial tissue damage, which produce an inflammatory neutrophil response. Upon dysregulation of these responses, inflammation, further tissue damage, and systemic spread of the pathogen may occur. Subsequent vascular inflammation and activation of coagulation processes may cause microvascular obstruction at sites distal to the primary site of infection. Low immunoglobulin (Ig) M and IgG levels have been detected in patients with severe infections like sCAP and sepsis, associated with increased severity and mortality. Based on Ig's modes of action, supplementation with polyvalent intravenous Ig preparations (standard IVIg or IgM/IgA-enriched Ig preparations) has long been discussed as a treatment option for severe infections. A prerequisite seems to be the timely administration of Ig preparations before excessive tissue damage has occurred and coagulopathy has developed. This review focuses on nonclinical and clinical studies that evaluated tissue-protective activities resulting from interactions of Igs with neutrophils, complement, and the coagulation system. The data indicate that coagulopathy, organ failure, and even death of patients can possibly be prevented by the timely combined interactions of (natural) IgM, IgA, and IgG with neutrophils and complement.

Atorvastatin protects against cyclophosphamide-induced thyroid injury in rats via modulation of JNK/ ERK/ p38 MAPK signaling pathway

BackgroundCancer chemotherapy is associated with various tissue toxicities that limit its use. Cyclophosphamide (CYC) is one of the most commonly used antineoplastic and immunosuppressive agent. Thyroid dysfunction is a critical side effect of anticancer drugs. Atorvastatin (ATV) is antihyperlipedemic drug with different tissue protective activities. The aim of this study was to determine the potential protective effect of ATV against CYC-induced thyroid injury in rats. MethodsATV was administered in the presence and absence of CYC. Thirty-two adult Wistar rats were randomly divided into four groups: control group, ATV group (20 mg/kg/day, p.o. for 14 day), CYC group (200 mg/kg, i.p. on day 9) and ATV/CYC group. Triiodothyronine (T3), thyroxine (T4), reduced glutathione (GSH), malondialdehyde (MDA), total nitrite/nitrate (NOx), p38 mitogen-activated protein kinase (P38MAPK), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK) were measured. In addition, thyroid histopathology and caspase 3 immunohistochemistry were performed. ResultsCYC significantly increased thyroid MDA, NOx, P38MAPK, ERK and JNK with decrease in GSH, T3 and T4 levels. Histopathological features of thyroid lesions and increased caspase 3 immune expression were appeared. ATV significantly normalized distributed oxidative, inflammatory and apoptotic indicators, resulting in an improvement of histopathological features and reduction of caspase 3 immunoexpression. ConclusionThese findings suggest that ATV protects against CYC-induced thyroid injury by regulating the JNK/ERK/p38-MAPK signaling pathway.

Graviola Protects Against Hepatic Toxicity Associated with DMBA induced Breast Cancer via Restoration of Antioxidants and Attenuation of Inflammatory Pathways

Aims: This study documented hepatic tissue's protective activity against oxidative damage mediated experimental models of breast cancer. Background: Gaviola has a long history of improving the protection mechanism against many diseases as an antioxidant and anticancer dietary agent. Objective: The purpose of this study is to establish changes in hepatic profiling, antioxidants, inflammatory cytokine expression, and DMBA-induced hepatic histopathology of mammalian rats. Methods: 7,12-dimethylbenz[a] anthracene (DMBA), PAHs, used orally in female Sprague Dawley rats Fifty-seven days-old with breast cancer single-dose diluted in sesame oil of 20 mg/kg/body weight. The cancer-bearing animals had 45 days gastrogavagated at 200 mg/kg/body weight with Graviola. The serum samples were taken at the end of the experiment. The rats were sacrificed to establish the hepatic protective activity of the Graviola by testing hepatic and oxidative stress markers. Results: Graviola therapy shows that enzymatic and non-enzymatic antioxidants and lipid peroxide levels have increased efficiency and have restored the high activity of hepatic marker enzymes such as ALT, AST, ALP, and GGT. To date, hepatic expression of nuclear factor erythroid 2-like 2 (Nfe2l2) and nuclear factor kappa B subunit 1 (Nfkb1) mediated rats have normalized. In addition, histological observations have demonstrated that Graviola's treatment effectively protects the liver from the oxidative damage caused by DMBA, reinforcing its hepatic defensive nature. Conclusion: Graviola therapy improves the efficiency of both enzymatic and non-enzymatic antioxidants and lipid peroxide levels. It also helps in the restoration of other liver enzymes.

TNF and TNF receptors as therapeutic targets for rheumatic diseases and beyond.

The cytokine TNF signals via two distinct receptors, TNF receptor 1 (TNFR1) and TNFR2, and is a central mediator of various immune-mediated diseases. Indeed, TNF-neutralizing biologic drugs have been in clinical use for the treatment of many inflammatory pathological conditions, including various rheumatic diseases, for decades. TNF has pleiotropic effects and can both promote and inhibit pro-inflammatory processes. The integrated net effect of TNF in vivo is a result of cytotoxic TNFR1 signalling and the stimulation of pro-inflammatory processes mediated by TNFR1 and TNFR2 and also TNFR2-mediated anti-inflammatory and tissue-protective activities. Inhibition of the beneficial activities of TNFR2 might explain why TNF-neutralizing drugs, although highly effective in some diseases, have limited benefit in the treatment of other TNF-associated pathological conditions (such as graft-versus-host disease) or even worsen the pathological condition (such as multiple sclerosis). Receptor-specific biologic drugs have the potential to tip the balance from TNFR1-mediated activities to TNFR2-mediated activities and enable the treatment of diseases that do not respond to current TNF inhibitors. Accordingly, a variety of reagents have been developed that either selectively inhibit TNFR1 or selectively activate TNFR2. Several of these reagents have shown promise in preclinical studies and are now in, or approaching, clinical trials.

1,5-Dicaffeoylquinic acid from Pseudognaphalium affine ameliorates dry eye disease via suppression of inflammation and protection of the ocular surface

PurposePseudognaphalium affine (P. affine), a medicinal plant, has long been used to treat various diseases due to its astringent and vulnerary effects. These therapeutic benefits are largely attributed to high contents of phytochemicals, such as flavonoids and polyphenols, that have anti-inflammatory and tissue-protective activities. Herein, we investigated the potential of dicaffeoylquinic acids (diCQAs), polyphenols from P. affine, as a novel treatment for dry eye disease (DED). MethodsWe isolated 1,5-, 3,4-, 3,5- and 4,5-diCQAs from the P. affine methanol extract, and tested the effects of diCQA isomers in cultures of human corneal epithelial cells (CECs) under desiccating hyperosmolar stress and in two mouse models for DED: desiccating environmental stress-induced DED and the NOD.B10-H2b mouse model of ocular Sjögren's syndrome. ResultsInitial screening showed that, among the diCQAs, 1,5-diCQA significantly inhibited apoptosis and enhanced viability in cultures of CECs under hyperosmolar stress. Moreover, 1,5-diCQA protected CECs by promoting proliferation and downregulating inflammatory activation. Subsequent studies with two mouse models of DED revealed that topical 1,5-diCQA administration dose-dependently decreased corneal epithelial defects and increased tear production while repressing inflammatory cytokines and T cell infiltration on the ocular surface and in the lacrimal gland. 1,5-diCQA was more effective in alleviating DED, as compared with two commercially-available dry eye treatments, 0.05% cyclosporine and 0.1% sodium hyaluronate eye drops. ConclusionsTogether, our results demonstrate that 1,5-diCQA isolated from P. affine ameliorates DED through protection of corneal epithelial cells and suppression of inflammation, thus suggesting a novel DED therapeutic strategy based on natural compounds.

Erythropoietin mediates re-programming of endotoxin-tolerant macrophages through PI3K/AKT signaling and protects mice against secondary infection.

Initial lipopolysaccharide (LPS) exposure leads to a hypo-responsive state by macrophages to a secondary stimulation of LPS, known as endotoxin tolerance. However, recent findings show that functions of endotoxin-tolerant macrophages are not completely suppressed, whereas they undergo a functional re-programming process with upregulation of a panel of molecules leading to enhanced protective functions including antimicrobial and tissue-remodeling activities. However, the underlying molecular mechanisms are still elusive. Erythropoietin (EPO), a glycoprotein regulated by hypoxia-inducible factor 1α (HIF-1α), exerts anti-inflammatory and tissue-protective activities. Nevertheless, the potential effects of EPO on functional re-programming of endotoxin-tolerant macrophages have not been investigated yet. Here, we found that initial LPS exposure led to upregulation of HIF-1α/EPO in macrophages and that EPO enhanced tolerance in tolerized macrophages and mice as demonstrated by suppressed proinflammatory genes such as Il1b, Il6, and Tnfa after secondary LPS stimulation. Moreover, we showed that EPO improved host protective genes in endotoxin-tolerant macrophages and mice, such as the anti-bacterial genes coding for cathelicidin-related antimicrobial peptide (Cnlp) and macrophage receptor with collagenous structure (Marco), and the tissue-repairing gene vascular endothelial growth factor C (Vegfc). Therefore, our findings indicate that EPO mediates the functional re-programming of endotoxin-tolerant macrophages. Mechanistically, we found that PI3K/AKT signaling contributed to EPO-mediated re-programming through upregulation of Irak3 and Wdr5 expression. Specifically, IL-1 receptor-associated kinase 3 (IRAK3) was responsible for inhibiting proinflammatory genes Il1b, Il6, and Tnfa in tolerized macrophages after LPS rechallenge, whereas WDR5 contributed to the upregulation of host beneficial genes including Cnlp, Marco, and Vegfc. In a septic model of mice, EPO pretreatment significantly promoted endotoxin-tolerant re-programming, alleviated lung injury, enhanced bacterial clearance, and decreased mortality in LPS-tolerized mice after secondary infection of Escherichia coli. Collectively, our results reveal a novel role for EPO in mediating functional re-programming of endotoxin-tolerant macrophages; thus, targeting EPO appears to be a new therapeutic option in sepsis and other inflammatory disorders.

MCTR3 reprograms arthritic monocytes to upregulate Arginase-1 and exert pro-resolving and tissue-protective functions in experimental arthritis.

SummaryBackgroundRheumatoid arthritis (RA) is a progressive degenerative disorder that leads to joint destruction. Available treatments only target the inflammatory component with minimal impact on joint repair. We recently uncovered a previously unappreciated family of pro-resolving mediators, the maresin conjugate in tissue regeneration (MCTR), that display both immunoregulatory and tissue-protective activities. Thus, we queried whether the production of these autacoids is disrupted in RA patients and whether they can be useful in treating joint inflammation and promoting joint repair.MethodsUsing a highly phenotyped RA cohort we evaluated plasma MCTR concentrations and correlated these to clinical markers of disease activity. To evaluate the immunoregulatory and tissue reparative activities we employed both in vivo models of arthritis and organ culture models.FindingsHerein, we observed that plasma MCTR3 concentrations were negatively correlated with joint disease activity and severity in RA patients. Evaluation of the mechanisms engaged by this mediator in arthritic mice demonstrated that MCTR3 reprograms monocytes to confer enduring joint protective properties. Single cell transcriptomic profiling and flow cytometric evaluation of macrophages from mice treated with MCTR3-reprogrammed monocytes revealed a role for Arginase-1 (Arg-1) in mediating their joint reparative and pro-resolving activities. Arg-1 inhibition reversed both the anti-arthritic and tissue reparative actions of MCTR3-reprogrammed monocytes.InterpretationOur findings demonstrate that circulating MCTR3 levels are negatively correlated with disease in RA. When administered to mice in vivo, MCTR3 displayed both anti-inflammatory and joint reparative activities, protecting both cartilage and bone in murine arthritis. These activities were, at least in part, mediated via the reprogramming of mononuclear phagocyte responses.FundingThis work was supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant no: 677542) and the Barts Charity (grant no: MGU0343) to J.D. J.D. is also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 107613/Z/15/Z).

Tissue-protective activity of selenomethionine and D-panthetine in B16 melanoma-bearing mice under doxorubicin treatment is not connected with their ROS scavenging potential.

AimTo evaluate molecular mechanisms of tissue-protective effects of antioxidants selenomethionine (SeMet) and D-pantethine (D-Pt) applied in combination with doxorubicin (Dx) in B16 melanoma-bearing-mice.MethodsImpact of the chemotherapy scheme on a survival of tumor-bearing animals, general nephro- and hepatotoxicity, blood cell profile in vivo, and ROS content in B16 melanoma cells in vitro was compared with the action of Dx applied alone. Nephrotoxicity of the drugs was evaluated by measuring creatinine indicator assay, hepatotoxicity was studied by measuring the activity of ALT/AST enzymes, and myelotoxicity was assessed by light microscopic analysis of blood smears. Changes in ROS content in B16 melanoma cells under Dx, SeMet, and D-Pt action in vitro were measured by incubation with fluorescent dyes dihydrodichlorofluoresceindiacetate (DCFDA, H2O2-specific) and dihydroethidium (DHE, O2--specific), and further analysis at FL1 (DCFDA) or FL2 channels (DHE) of FACScan flow cytometer. The impact of aforementioned compounds on functional status of mitochondria was measured by Rhodamine 123 assay and further analysis at FL1 channel of FACScan flow cytometer.ResultsSelenomethionine (1200 µg/kg) and D-pantethine (500 mg/kg) in combination with Dx (10 mg/kg) significantly reduced tumor-induced neutrophilia, lymphocytopenia, and leukocytosis in comparison to Dx treatment alone. Moreover, SeMet and D-Pt decreased several side effects of Dx, namely an elevated creatinine level in blood and monocytosis, thus normalizing health conditions of B16 melanoma-bearing animals.ConclusionsOur results showed that antioxidants selenomethionine and D-pantethine possess significant nephroprotective and myeloprotective activity toward Dx action on murine B16 melanoma in vivo, but fail to boost a survival of B16 melanoma-bearing animals. The observed cytoprotective effects of studied antioxidants are not directly connected with their ROS scavenging.

Lesional accumulation of CD8(+) cells in sciatic nerves of experimental autoimmune neuritis rats.

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies. Macrophages are the major immune cells in peripheral nerves and may exert tissue-damage or tissue-protective activity during EAN. While considered to define a subpopulation of T lymphocytes, CD8 expression has been found on certain macrophages that show cytotoxic effects. Here we have studied the spatiotemporal accumulation of CD8(+) cells in sciatic nerves of EAN rats. A robust accumulation of CD8(+) cells was observed in the sciatic nerves of EAN rats, which was positively correlated with the severity of neurological signs in EAN. Moreover, double-labelling experiments showed that the major cellular sources of CD8 were reactive macrophages. Therefore, our data here suggest a pathological role of CD8(+) macrophages in EAN, which makes CD8(+) macrophage a potential therapeutic target for EAN.

Evaluation of pharmacodynamic activities of EPs® 7630, a special extract from roots of Pelargonium sidoides, in animals models of cough, secretolytic activity and acute bronchitis

BackgroundEPs® 7630 is a proprietary aqueous-ethanolic extract from roots of Pelargonium sidoides DC and has been demonstrated to dispose among others of antibacterial, antiviral, immunomodulatory, antioxidant, and tissue-protective activity. It is an approved medicinal product in more than 50 countries for the treatment of airway infections such as acute bronchitis, common cold, and sinusitis. PurposeWhile the pharmacological effects of EPs® 7630 have extensively been evaluated in diverse in vitro test systems, the number of publications reporting results from in vivo models is limited. Study designIn the present study antitussive, secretolytic, and anti-inflammatory effects of EPs® 7630 were assessed in animal experiments following oral administration at human equivalent doses. MethodsAntitussive effects were evaluated using ammonia- and citric acid-induced models of cough in mice (20, 40, 120 mg/kg) and guinea pigs (10, 20, 45 mg/kg), respectively. For the determination of secretolytic activity tracheobronchial secretion of intraperitoneally injected phenol red was determined in mice, while antiinflammatory action was assessed in an acute bacterial bronchitis model in rats. ResultsA significant and dose-dependent reduction of cough frequency was observed in both cough models, which was accompanied by a prolongation of cough latency time. Similarly, the extract exerted a marked secretolytic activity in mice. Induction of acute bacterial bronchitis caused characteristic histopathological changes in lung tissue adjacent to trachea and bronchi. The degree of these lesions was significantly reduced in rats treated with EPs® 7630 at doses of 30 and 60 mg/kg. This protective effect at least partially seems to be mediated by an up-regulation of superoxide dismutase and a subsequent protective effect against oxidative stress as indicated by a reduced serum level of malondialdehyde. ConclusionThe present data further support the therapeutic use of EPs® 7630 in respiratory tract infections and provide a basis for detailed studies on its bioactive constituents as well as their in vivo mode of action.

Asialoerythropoietin ameliorates bleomycin-induced acute lung injury in rabbits by reducing inflammation.

Acute lung injury, a critical illness characterized by acute respiratory failure with bilateral pulmonary infiltrates, remains unresponsive to current treatments. The condition involves injury to the alveolar capillary barrier, neutrophil accumulation and the induction of proinflammatory cytokines followed by lung fibrosis. In the present study, a rabbit model of bleomycin-induced acute lung injury was established to examine the effects of asialoerythropoietin (AEP), an agent with tissue-protective activities, on pulmonary inflammation. Six Japanese white rabbits were randomly divided into two equal groups. Acute lung injury was induced in all rabbits by intratracheally injecting bleomycin. The control group was injected with bleomycin only; the experimental (AEP) group was injected intravenously with AEP (80 μg/kg) prior to the bleomycin injection. Computed tomography (CT) studies were performed seven days later. The CT inflammatory scores of areas exhibiting abnormal density and the pathological inflammatory scores were recorded as a ratio on a 7×7 mm grid. The CT and pathological inflammatory scores were significantly different between the control and AEP groups [122±10 and 16.3±1.5 (controls) vs. 71±8.5 and 9.7±1.4 (AEP), respectively; P<0.01]. Thus, the present study revealed that AEP prevents bleomycin-induced acute lung injury in rabbits.

Definition of a Family of Tissue-Protective Cytokines Using Functional Cluster Analysis: A Proof-of-Concept Study

The discovery of the tissue-protective activities of erythropoietin (EPO) has underlined the importance of some cytokines in tissue-protection, repair, and remodeling. As such activities have been reported for other cytokines, we asked whether we could define a class of tissue-protective cytokines. We therefore explored a novel approach based on functional clustering. In this pilot study, we started by analyzing a small number of cytokines (30). We functionally classified the 30 cytokines according to their interactions by using the bioinformatics tool STRING (Search Tool for the Retrieval of Interacting Genes), followed by hierarchical cluster analysis. The results of this functional clustering were different from those obtained by clustering cytokines simply according to their sequence. We previously reported that the protective activity of EPO in a model of cerebral ischemia was paralleled by an upregulation of synaptic plasticity genes, particularly early growth response 2 (EGR2). To assess the predictivity of functional clustering, we tested some of the cytokines clustering close to EPO (interleukin-11, IL-11; kit ligand, KITLG; leukemia inhibitory factor, LIF; thrombopoietin, THPO) in an in vitro model of human neuronal cells for their ability to induce EGR2. Two of these, LIF and IL-11, induced EGR2 expression. Although these data would need to be extended to a larger number of cytokines and the biological validation should be done using more robust in vivo models, rather then just one cell line, this study shows the feasibility of this approach. This type of functional cluster analysis could be extended to other fields of cytokine research and help design biological experiments.

Application of Interleukin-22 Mediates Protection in Experimental Acetaminophen-Induced Acute Liver Injury

Acetaminophen (APAP, paracetamol)-induced hepatotoxicity, although treatable by timely application of N-acetylcysteine, can be fatal. Because it is among the common causes of acute liver failure in intensive care units and in light of its gradually increasing incidence, the need for novel therapeutic strategies aimed at severe intoxication is apparent. Recently, it has been shown that IL-22, a STAT3-activating cytokine, has the capability to mediate liver protection. Herein, the protective potential of IL-22 in murine APAP-induced hepatotoxicity was assessed. Intravenous administration of prophylactic IL-22 significantly reduced serum alanine aminotransferase levels and histopathologic damage in APAP-induced liver injury, a process that coincided with increased hepatocyte proliferation invivo. Concomitant gene expression analysis revealed hepatic induction of genes prototypically up-regulated by the IL-22/STAT3 axis, among others suppressor of cytokine signaling-3, lipocalin-2, and α1-antichymotrypsin. Notably, in a translational setting of therapeutic treatment 2 hours after APAP, IL-22 supported protection in the context of suboptimal N-acetylcysteine dosing. IL-22 likewise connected to augmented hepatocyte proliferation in this experimental setting. As detected by analysis of inflammatory cytokine production, systemically applied IL-22 did not display acute immunomodulation/stimulation in otherwise untreated or endotoxemic mice. Those latter observations clearly confirm acute tolerability of systemically applied IL-22. Observations presented altogether suggest that therapeutic IL-22 administration is a conceivable tissue-protective regimen aimed at hard-to-treat patients with severe APAP-induced hepatotoxicity.

PS194. Role of Erythropoietin Receptors and Ligands in Attenuating Inflammation and Apoptosis in Critical Limb Ischemia

Objectives: Erythropoietin (EPO) exerts tissue-protective effect through a hetero-receptor complex (EPORCD131). The aims of this study were to: 1. Examine the expression of EPOR-CD131 in skeletal muscle obtained from human critical limb ischaemia (CLI) compared with control muscle; and 2. Investigate the tissue protective effects of EPO and a non-haematopoietic helix-B peptide of EPO (ARA 290) in an in vitro model of skeletal myotube ischemia. Methods: Tissue samples were obtained from the gastrocnemius muscle of patients undergoing perigenicular lower extremity amputation for CLI (n 12). Control samples were obtained from patients undergoing cardiac surgery (n 12). Immunohistochemistry and western blot were used to demonstrate the expression of EPOR-CD131. An in vitro model system of skeletal myotube ischemia was created using C2C12 myotubes in hypoxic chambers. Myotubes were pre-treated with EPO or ARA 290 before exposure to simulated ischemia. Inflammatory cytokines were measured using ELISA. Apoptosis and cell death were determined by cleaved caspase-3 assay and lactate dehydrogenase (LDH) release respectively. Results: There was an expression of EPOR and CD131 in both ischemic and normal skeletal muscle cells with co-localization of EPOR-CD131. The expression of receptors was upregulated in CLI (p 0.01). EPO and ARA 290 attenuated the release of Interleukin-6, and reduced the cleaved caspase-3 and the release of LDH from the myotubes exposed to simulated ischemia (p 0.01). Conclusions: Here we report the first study demonstrating expression of EPO receptors within skeletal muscle cells and elevated expression in CLI. We also show that EPO, and the non-haematopoietic helix-B peptide of EPO (ARA 290), decreases inflammation and apoptosis of ischemic myotubes in vitro. Our data suggests that the use of EPO derivatives (ARA 290) to selectively enhance the tissue protective activity of EPO may provide a novel therapeutic avenue for CLI.

Non-erythropoietic tissue-protective peptides derived from erythropoietin: WO2009094172

Erythropoietin (EPO) is a cytokine with erythropoietic and tissue protective activities. Its action as a tissue protective agent requires, however, high dosage that results in limiting side effects associated with abnormally augmented erythropoiesis. Elimination of the erythropoietic activity of EPO while preserving its tissue protective properties was nevertheless achieved in carbamoylated EPO (CEPO), whose therapeutic activity and apparent safety were documented in experimental models of nervous, heart, kidney and other tissue damage, justifying ongoing clinical trials. Here, we review patent application WO2009094172 by Araim Pharmaceuticals, which describes novel EPO-derived peptides having tissue protective but no erythropoietic activity. The preferred peptide, UEQLERALNSS, which mimics the external 3D structure of the helix B of EPO, was shown to exhibit the same spectrum of tissue protective activity as CEPO in several in vivo models. In addition, it could reduce radiation-induced mortality when administered 24 h after irradiation in mice, suggesting its possible utility in emergency situations after mass irradiation casualties. Owing to their low manufacturing cost, high stability and low immunogenicity, such peptides might well offer a superior alternative to CEPO for therapeutic tissue protection in human pathologies and are likely to provide valuable probes to study the molecular mechanisms of EPO-mediated cytoprotection.

Evaluation of Tissue Protective Activity of Tinospora cordifolia Stems in Irradiated Swiss Albino Mice with 60 Co Radiation

In the present study, wholesome tissue protective efficiency of the ethanolic extract of Tinospora cordifolia stems in mice in terms of whole body survival, genotoxicity, cell proliferation and hematological profile have been investigated. The animals were divided into four groups having six animals in each. Group I, (Control), received vehicle, Group II received orally ethanolic extract of Tinospora cordifolia (500mg/kg), Group III was irradiated with 60 Co radiation (external gamma radiation of 4 GY for 24h), and animals of Group IV were irradiated with 60 Co radiation (external gamma radiation of 4 GY for 24 h) and given orally ethanolic extract of Tinospora cordifolia (500mg/kg), for seven days. On the 8th day all the animals were sacrificed, the blood was collected and hematological cell profile was analyzed. Intestine was removed and its histopathology was done and their mitotic index and crypt of villi was counted. The drug shows significant tissue protective activity (P<0.05) and shows normalization in hematological cell profile in irradiated animals.

Carbamylated darbepoetin derivative prevents endothelial progenitor cell damage with no effect on angiogenesis

Erythropoietin (EPO) prevents cell apoptosis induced by oxidative stress. Carbamylated EPO maintains the tissue-protective activities of the unmodified EPO but does not stimulate erythropoiesis. This study evaluates whether carbamylated erythropoietin is as effective as recombinant human erythropoietin in protecting endothelial progenitor cells (EPCs) from apoptosis without stimulating erythropoiesis. Experiments were performed in an erythroid cell line (UT-7) and in human EPCs. Cell signals regulating proliferation and apoptosis (Jak-2, Akt, Erk1/2, NFκB and Stat-5) were measured by Western blotting. In human EPCs, cell senescence, apoptosis and proliferation were assessed by acidic β-gal and measurement of telomere length, TUNEL and PCNA labeling, respectively. Angiogenesis was evaluated using the endothelial tube formation assay. In UT-7, carbamylated erythropoietin (C-darbe) induced phosphorylation of the anti-apoptotic Jak-2/Akt signal and, as opposed to recombinant human erythropoietin (darbe), did not produce a significant activation of cell proliferating signals. Darbe increased the percent of proliferating EPCs and promoted angiogenesis. By contrast, C-darbe failed to stimulate proliferation of EPCs. Both C-darbe and darbe equally reduced apoptosis and senescence. Thus, C-darbe protects EPCs from apoptosis and does not increase erythropoiesis.

Does the concomitant intra-arterial injection of asialoerythropoietin and edaravone mitigate ischaemic mucosal damage after acute superior mesenteric artery thromboembolism in a rabbit autologous fibrin clot model?

To increase the survival rate of patients with acute superior mesenteric artery thromboembolism (ASMAT) treated by catheter thrombolysis, we examined the effects of delivering edaravone and asialoerythropoietin, agents with tissue-protective activities, using a rabbit autologous fibrin clot ASMAT model. Japanese white rabbits (n=32) were randomly separated into four equal groups. 45 min after introducing autologous fibrin clot, Group U received urokinase and heparin; Group E received urokinase and heparin plus edaravone; Group A received urokinase and heparin plus asialoerythropoietin; and Group EA received urokinase, heparin and edaravone plus asialoerythropoietin via a catheter. The intestines were removed 6 h later and intestinal mucosal damage was scored using the Park's injury score. Survival time was assessed. Average mucosal injury was 5.78+/-1.52 (Group U), 2.88+/-0.72 (Group E), 1.90+/-1.23 (Group A) and 1.18+/-1.25 (Group EA). The degree of mucosal injury was significantly lower in Group EA than in Groups U and E (p<0.05). Conversely, there was no significant difference between Group A and Group EA, or between Group A and Group E. The survival times were 31.50+/-13.30 h (Group U), 51.00+/-24.74 h (Group E), 48.00+/-16.97 h (Group A) and 82+/-51.07 h (Group EA); the difference among the four groups was not significant. In conclusion, the concomitant administration of asialoerythropoietin and edaravone reduced mucosal membrane injury significantly compared with edaravone alone. However, to improve the survival of ASMAT rabbit models, the delivery of an appropriate dose of asialoerythropoietin is required, together with the development of methods to assess peripheral recanalisation.

Plant recombinant erythropoietin attenuates inflammatory kidney cell injury

Human erythropoietin (EPO) is a pleiotropic cytokine with remarkable tissue-protective activities in addition to its well-established role in red blood cell production. Unfortunately, conventional mammalian cell cultures are unlikely to meet the anticipated market demands for recombinant EPO because of limited capacity and high production costs. Plant expression systems may address these limitations to enable practical, cost-effective delivery of EPO in tissue injury prevention therapeutics. In this study, we produced human EPO in tobacco and demonstrated that plant-derived EPO had tissue-protective activity. Our results indicated that targeting to the endoplasmic reticulum (ER) provided the highest accumulation levels of EPO, with a yield approaching 0.05% of total soluble protein in tobacco leaves. The codon optimization of the human EPO gene for plant expression had no clear advantage; furthermore, the human EPO signal peptide performed better than a tobacco signal peptide. In addition, we found that glycosylation was essential for the stability of plant recombinant EPO, whereas the presence of an elastin-like polypeptide fusion had a limited positive impact on the level of EPO accumulation. Confocal microscopy showed that apoplast and ER-targeted EPO were correctly localized, and N-glycan analysis demonstrated that complex plant glycans existed on apoplast-targeted EPO, but not on ER-targeted EPO. Importantly, plant-derived EPO had enhanced receptor-binding affinity and was able to protect kidney epithelial cells from cytokine-induced death in vitro. These findings demonstrate that tobacco plants may be an attractive alternative for the production of large amounts of biologically active EPO.

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin

Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the beta common receptor. In the present work, we have delimited tissue-protective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58-82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia-reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPO's trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo. Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPO's three-dimensional structure.