Cardenolide and pregnatriene compounds from the roots of Nerium oleander.

Cardenolide and pregnatriene compounds were isolated from the chloroform fraction of the 95% aqueous ethanolic extract of dried roots of Nerium oleander. The stereochemical structure of the cardenolide and pregnatriene compounds was determined to be 3ß-O-(D-diginosyl)-14ß-hydroxy card-20(22)-enolide and 12ß-hydroxy pregna-4,6,16-triene-3,20-dione using spectroscopic methods including IR, HRMS and NMR spectroscopy.

Structure of the SARS-CoV-2 Nsp1/5'-Untranslated Region Complex and Implications for Potential Therapeutic Targets, a Vaccine, and Virulence.

SARS-CoV-2 is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic around the world causing pneumonia and lower respiratory tract infections. In understanding the SARS-CoV-2 pathogenicity and mechanism of action, it is essential to depict the full repertoire of expressed viral proteins. The recent biological studies have highlighted the leader protein Nsp1 of SARS-CoV-2 importance in shutting down the host protein production. Besides, it still enigmatic how Nsp1 regulates for translation. Here we report the novel structure of Nsp1 from SARS-CoV-2 in complex with the SL1 region of 5'UTR of SARS-CoV-2, and its factual interaction is corroborated with enzyme kinetics and experimental binding affinity studies. The studies also address how leader protein Nsp1 of SARS-CoV-2 recognizes its self RNA toward translational regulation by further recruitment of the 40S ribosome. With the aid of molecular dynamics and simulations, we also demonstrated the real-time stability and functional dynamics of the Nsp1/SL1 complex. The studies also report the potential inhibitors and their mode of action to block viral protein/RNA complex formation. This enhance our understanding of the mechanism of the first viral protein Nsp1 synthesized in the human cell to regulate the translation of self and host. Understanding the structure and mechanism of SARS-CoV-2 Nsp1 and its interplay with the viral RNA and ribosome will open the arena for exploring the development of live attenuated vaccines and effective therapeutic targets for this disease.

The Effects of Ascorbic Acid and U-74389G on Renal Ischemia-Reperfusion Injury in a Rat Model.

BACKGROUND/AIM: U-74389G and ascorbic acid protect the cells from oxidation. This study aimed to depict their role in ischemia-reperfusion injury in a renal rat model. MATERIALS AND METHODS: Sixty Wistars rats were randomized into six groups of 10 animals each. Group A Ischemia 30 min, reperfusion 60 min; Group B Ischemia 30 min, reperfusion 120 min; Group C Ischemia 30 min, ascorbic acid administration, reperfusion 60 min; Group D Ischemia 30 min, ascorbic acid administration, reperfusion 120 min; Group E Ischemia 30 min, U-74389G administration, reperfusion 60 min; Group F Ischemia 30 min, U-74389G administration, reperfusion 120 min. We then collected tissue and blood samples. RESULTS: Histology and the significantly decreased malondialdehyde and tumor necrosis factor-α levels indicated that ascorbic acid was superior to U-74389G, at pre-defined time intervals. CONCLUSION: Ascorbic acid and U-74389G ameliorated renal damage induced by ischemia-reperfusion injury, suggesting a therapeutic effect.

Revisiting the action of steroids and triterpenoids on the human sperm Ca2+ channel CatSper.

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) is vital for male fertility. Contradictory findings have been published on the regulation of human CatSper by the endogenous steroids estradiol, testosterone and hydrocortisone, as well as the plant triterpenoids, lupeol and pristimerin. The aim of this study was to elucidate this controversy by investigating the action of these steroids and plant triterpenoids on human CatSper using population-based Ca2+-fluorimetric measurements, the specific CatSper-inhibitor RU1968 and a functional test assessing the CatSper-dependent penetration of human sperm cells into methylcellulose. Estradiol, testosterone and hydrocortisone were found to induce Ca2+-signals in human sperm cells with EC50 values in the lower µM range. By employing the specific CatSper-inhibitor RU1968, all three steroids were shown to induce Ca2+-signals through an action on CatSper, similar to progesterone. The steroids were found to dose-dependently inhibit subsequent progesterone-induced Ca2+-signals with IC50 values in the lower µM range. Additionally, the three steroids were found to significantly increase the penetration of human sperm cells into methylcellulose, similar to the effect of progesterone. The two plant triterpenoids, lupeol and pristimerin, were unable to inhibit progesterone-induced Ca2+-signals, whereas the CatSper-inhibitor RU1968 strongly inhibited progesterone-induced Ca2+-signals. In conclusion, this study supports the claim that the steroids estradiol, testosterone and hydrocortisone act agonistically on CatSper in human sperm cells, thereby mimicking the effect of progesterone, and that lupeol and pristimerin do not act as inhibitors of human CatSper.

Experimental isolation and preservation of solid organs before transplantation: effects of pretreatment using four different molecules.

OBJECTIVES: In transplantation surgery, the ischaemic organ and reperfusion impairment after cold storage remains a considerable risk factor for impaired function and potential failure of the grafted organ. Substantial logistical efforts have been undertaken to reduce the cold ischaemic time because the demand for available transplant organs and the periods of cold ischaemia are increasing. METHODS: Four molecules were investigated (erythropoietin, sildenafil, lazaroid [U74389G], octreotide) in individual intravenous infusions 1 hour before the organ was harvested. This study was performed in 30 healthy landrace/large-white pigs (male; >10 weeks old; average weight, 22 ± 2 kg) in groups of six. The organs were studied at harvest, and at 8 and 24 hours post-harvest. RESULTS: The lazaroid molecule increased malondialdehyde (MDA) levels in the liver and pancreas at 8 hours. Hepatic lazaroid molecules improved liver histology at 8 and 24 hours. For kidneys, erythropoietin had a positive effect at 24 hours post-harvest. For the pancreas, octreotide showed better performance. In the lungs, there was less interstitial oedema with erythropoietin and lazaroid compared with the control group at 8 hours post-harvest. CONCLUSION: All molecules had a positive effect and decreased ischaemia/reperfusion graft injury. Thus, pretreatment before organ harvest has a beneficial role.

Pharmacological inhibition of lipid peroxidative damage by the 21-aminosteroid U-74389G improves cortical mitochondrial function following traumatic brain injury in young adult male rats.

The 21-aminosteroid ("lazaroid") U-74389G (U74), an inhibitor of lipid peroxidation (LP), was used to protect mitochondrial function following TBI in young adult male rats. The animals received a severe (2.2 mm) controlled cortical impact-TBI. U74 was administered intravenous at 15 min and 2 h post injury (hpi) followed by intraperitoneal dose at 8 hpi at the following doses (mg/kg): 0.3 (IV) + 1 (IP), 1 + 3, 3 + 10, 10 + 30. Total cortical mitochondria were isolated at 72 hpi and respiratory rates were measured. Mitochondrial 4-HNE and acrolein were evaluated as indicators of LP-mediated oxidative damage. At 72 h post-TBI injured animals had significantly lower mitochondrial respiration rates compared to sham. Administration of U74 at the 1 mg/kg dosing paradigm significantly improved mitochondrial respiration rates for States II, III, V(II) and RCR compared to vehicle-treated animals. At 72 h post-TBI injured animals administration of U74 also reduced reactive aldehydes levels compared to vehicle-treated animals. The aim of this study was to explore the hypothesis that interrupting secondary oxidative damage via acute pharmacological inhibition of LP by U74 following a CCI-TBI would provide mitochondrial neuroprotective effects in a dose-dependent manner. We found acute administration of U74 to injured rats resulted in improved mitochondrial function and lowered the levels of reactive aldehydes in the mitochondria. These results establish not only the most effective dose of U74 treatment to attenuate LP-mediated oxidative damage, but also set the foundation for further studies to explore additional neuroprotective effects following TBI.

Beneficial Effect of U-74389 G and Sildenafil in An Experimental Model of Flap Ischemia/Reperfusion Injury in Swine. Histological and Biochemical Evaluation of the Model.

Purpose of the study: Tissue reconstruction after burns, tumor excisions, infections or injuries is a frequent surgical challenge to avoid Ischemia-reperfusion injury. Lazaroids and sildenafil, through their mechanisms of action, have been studied for their protective effects on various organs subjected to IRI. In this study, we aimed to evaluate the therapeutic potential of U-74389G and sildenafil in a swine model of ischemia and reperfusion injury of latissimus dorsi flap. Materials and methods: Forty-two Landrace male pigs, weighing 28-35 kg, were equally (n = 6) randomized into the following groups: (a) Group I: control, (b) Group II: administration of U-74389G after ischemia, (c) Group III: administration of sildenafil after ischemia, (d) Group IV: administration of U-74389G and sildenafil after ischemia, (e) Group V: administration of U-74389G prior to ischemia, (f) Group VI: administration of sildenafil prior to ischemia, and (g) Group VII: administration of U-74389G and sildenafil prior to ischemia. Blood and tissue sampling was conducted before ischemia, 15 and 30 min after occlusion, 30, 60, 90, and 120 min after reperfusion. Results: Statistically significant reduction (p < 0.05) was detected in lymphocytes and polymorphonuclear leukocytes concentrations as well as in the appearance of edema after histopathologic evaluation of the ischemic tissue, especially in the groups of combined treatment. Measurements of malondialdeyde and tumour necrosis factor alpha in tissues revealed a significant decrease (p < 0.001) of these markers in the treatment groups when compared to the control, particularly in the latest estimated timepoints. Conclusions: The synergistic action of U-74389G and sildenafil seems protective and promising in cases of flap IRI during tissue reconstruction surgery.

3,20-Dihydroxy-13α-19-norpregna-1,3,5(10)-trienes. Synthesis, structures, and cytotoxic, estrogenic, and antiestrogenic effects.

New 3,20-dihydroxy-13α-19-norpregna-1,3,5(10)-trienes were synthesized. The effects of these compounds on breast cancer cells and ERα activation were investigated. The scaffold of compounds containing the six-membered ring D' annulated at 16α,17α-positions was constructed via the Lewis acid catalyzed Diels-Alder reaction of butadiene with 3-methoxy-13α-19-norpregna-1,3,5(10),16-tetraen-20-one 5 under a pressure of 600 MPa. The hydrogenation of primary cyclohexene adduct 6 followed by the one-pot reduction-demethylation (DIBAH) gave target epimeric 3,20-dihydroxy steroids 8a and 8b. The Corey-Chaykovsky reaction of the same conjugated ketone 5 gave a 16α,17α-methylene-substituted compound. The reaction of the latter with DIBAH yielded 3,20(R,S)-dihydroxy-16α,17α-methyleno-13α-19-norpregna-1,3,5(10)-triene 10. The hydrogenation of the 16,17-double bond of compound 5 produced a mixture of 17α- and 17ß-epimeric ketones, reduction-demethylation of which gave 3,20(S)-dihydroxy-13α,17α-19-norpregna-1,3,5(10)-triene 12a and 3,20(R)-dihydroxy-13α,17ß-19-norpregna-1,3,5(10)-triene 12b. All compounds were fully characterized by 1D and 2D NMR, HRMS, and X-ray diffraction. All target compounds showed pronounced cytotoxic effect against MCF-7 breast cancer cells and NCI/ADR-RES doxorubicin-resistant cells at micromolar concentrations. The ERα-mediated luciferase reporter gene assay demonstrated that all compounds, except for compound 10, are ERα inhibitors, while cyclopropane compound 10 proved to be an ERα activator. Docking experiments showed that all compounds are well accommodated to LBD ERα but have some differences in the binding mode.

Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion.

INTRODUCTION: Lung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. MATERIALS AND METHODS: A total of 24 pigs were randomized in three groups. Group I (n = 8) underwent sham operation, group II (n = 8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n = 8) received 3 doses of lazaroid (3 mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7 t h postoperative day and lung specimens were harvested for molecular analysis. RESULTS: mRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (-63.7%), LTC4S (-35.9%) and iNOS (-60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). CONCLUSION: Lazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.

A novel cross-species inhibitor to study the function of CatSper Ca2+ channels in sperm.

BACKGROUND AND PURPOSE: Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. EXPERIMENTAL APPROACH: We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. KEY RESULTS: RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. CONCLUSION AND IMPLICATIONS: We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species.

Enhanced brain delivery with lower hepatic exposure of lazaroid loaded nanostructured lipid carriers developed using a design of experiment approach.

The current study was designed to develop and optimize lazaroid loaded nano-structured lipid carriers (LAZ-NLCs) using design of experiment approach for enhancing lazaroid brain exposure. Response surface plots were used to determine the effects of independent variables (amount of PEGylating agent and liquid lipid) on dependent variables (particle size, zeta potential and encapsulation efficiency), while numerical optimization was used for optimizing LAZ-NLCs composition. The optimal LAZ-NLCs were spherical in shape with measured size of 172.3 ±â€¯3.54 nm, surface charge of -4.54 ±â€¯0.87 mV and encapsulation efficiency of 85.01 ±â€¯2.60%. The optimal LAZ-NLCs were also evaluated for hemolytic potential, storage stability and solid-state properties. The plasma pharmacokinetics along with brain and hepatic distributions of control lazaroid citrate solution and optimal LAZ-NLCs formulation were evaluated in Sprague-Dawley rats after the single bolus intravenous administration. The optimized LAZ-NLCs and the control lazaroid citrate solution had similar plasma pharmacokinetic profiles; however, differential organ bio-distributions were observed. The lazaroid exposure in brain was enhanced by two times with a decreased liver exposure by half for the NLCs group compared to the solution group.

Neurosurgeon academic impact is associated with clinical outcomes after clipping of ruptured intracranial aneurysms.

BACKGROUND: Surgeon-dependent factors such as experience and volume are associated with patient outcomes. However, it is unknown whether a surgeon's research productivity could be related to outcomes. The main aim of this study is to investigate the association between the surgeon's academic productivity and clinical outcomes following neurosurgical clipping of ruptured aneurysms. METHODS: We performed a post-hoc analysis of 3567 patients who underwent clipping of ruptured intracranial aneurysms in the randomized trials of tirilazad mesylate from 1990 to 1997. These trials included 162 centers and 156 surgeons from 21 countries. Primary and secondary outcomes were: Glasgow outcome scale score and mortality, respectively. Total publications, H-index, and graduate degrees were used as academic indicators for each surgeon. The association between outcomes and academic factors were assessed using a hierarchical logistic regression analysis, adjusting for patient covariates. RESULTS: Academic profiles were available for 147 surgeons, treating a total of 3307 patients. Most surgeons were from the USA (62, 42%), Canada (18, 12%), and Germany (15, 10%). On univariate analysis, the H-index correlated with better functional outcomes and lower mortality rates. In the multivariate model, patients under the care of surgeons with higher H-indices demonstrated improved neurological outcomes (p = 0.01) compared to surgeons with lower H-indices, without any significant difference in mortality. None of the other academic indicators were significantly associated with outcomes. CONCLUSION: Although prognostication following surgery for ruptured intracranial aneurysms primarily depends on clinical and radiological factors, the academic impact of the operating neurosurgeon may explain some heterogeneity in surgical outcomes.

Comparison of the Acute Hematopoietic Capacities of Erythropoietin and U-74389G Concerning Hematocrit Levels.

AIM: This study compared the hematopoietic capacities of erythropoietin (Epo) and antioxidant drug U-74389G, based on 2 preliminary studies. The provided results on hematocrit levels augmentation were co-evaluated in a hypoxia reoxygenation protocol of an animal model. MATERIALS AND METHODS: Hematocrit levels were evaluated at the 60th reoxygenation min (for groups A, C and E) and at the 120th reoxygenation min (for groups B, D and F) in 60 rats. Groups A and B received no drugs, rats from groups C and D were administered with Epo; whereas rats from groups E and F were administered with U-74389G. RESULTS: The first preliminary study of Epo non-significantly increased the hematocrit levels by 0.24%+1.38% (p-value=0.8586). The second preliminary study of U-74389G significantly raised the hematocrit levels by 3.16%+1.33% (p-value=0.0196). These 2 studies were co-evaluated since they came from the same experimental setting. The outcome of the co-evaluation was that U-74389G has approximately 12.66-fold higher hematopoietic potency than Epo (p-value=0.0000). CONCLUSION: The anti-oxidant capacities of U-74389G provide satisfactory acute hematopoietic properties; presenting approximately 12.66-fold hematocrit level rise than epo (p-value=0.0000).

Lazaroid U-74389G for cardioplegia-related ischemia-reperfusion injury: an experimental study.

BACKGROUND: The adverse effects of myocardial ischemia and reperfusion during cardiopulmonary bypass (CPB) have been thoroughly described. Lazaroid U-74389G, a 21 aminosteroid, has been shown to attenuate ischemia and reperfusion injury and improve recovery in a variety of experimental models. METHODS: Sixteen male swine were randomly divided in two groups. All animals underwent 45 min of ischemic cardioplegic arrest, with U-74389G addition to the standard cardioplegic solution, whereas controls underwent the same procedure without U-74389G. Creatine kinase-MB isoenzyme (CK-MB) and cardiac troponin T levels were measured immediately before CPB (time point 0), during the ischemic period (time point 1) and 30 (time point 2), 60 (time point 3), and 120 (time point 4) min after reperfusion. Myocardial biopsies were obtained at time points 0 and 4. RESULTS: CK-MB levels (in U/L) at time points 0-4 were 205 (186-235) versus 219 (196-269; P = 0.72), 215 (167-248) versus 253 (193-339; P = 0.23), 234 (198-255) versus 338 (249-441; P = 0.02), 244 (217-272) versus 354 (269-496; P = 0.01), and 285 (230-321) versus 439 (432-530; P < 0.01) in lazaroid-treated animals versus controls, respectively. Cardiac troponin T levels (in ng/L) at time points 0-4 were 58 (26-287) versus 237 (26-395; P = 0.72), 129 (61-405) versus 265 (145-525; P = 0.23), 261 (123-467) versus 474 (427-1604; P = 0.04), 417 (204-750) versus 841 (584-1818; P = 0.11), and 643 (353-1259) versus 1600 (1378-2313; P < 0.01), respectively. Necrosis grades at time point 4 were 0.0 (0.0-1.0) versus 1.5 (1.0-2.0; P < 0.01) in lazaroid-treated animals versus controls, respectively. CONCLUSIONS: The present study, in addition to reconfirming the well-described adverse effects of CPB, demonstrates the efficacy of the newer generation lazaroid U-74389G in alleviating these effects.

UPLC-MS/MS assay of 21-aminosteroid (lazaroid U74389G) for application in pharmacokinetic study.

Lazaroids are potent inhibitors of lipid peroxidation, both in vitro and in vivo. Additionally, a member of the lazaroid family, U-74389G (LAZ) has been shown to have specific radio-protective and anti-proliferative effects. However, there is no quantitative analytical method developed for measuring the therapeutic levels of LAZ for the aforementioned effects. This article highlights the development and validation of a sensitive UPLC-MS/MS method for the quantification of LAZ, and its subsequent application in pharmacokinetic studies in rats with the lower limit of quantification (LLOQ) of 1.95 ng/mL. LAZ and internal standard diadzein (IS) were separated using ACQUITY UPLC(®) BEH C18 column. Gradient elution was used at a flow rate of 0.45 mL/min with mobile phases consisting of 0.1% formic acid in water and 0.1% formic in acetonitrile. LAZ (m/z 612→260) and IS (m/z 255→199) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode on QTRAP(®) 5500 System. The UPLC-MS/MS method was validated as per the US FDA Guidelines for Bio-analytical Validation. LAZ was extracted from rat plasma (100 µL) using protein precipitation by acetonitrile with mean recovery and matrix factor in range of 47.7-56.1%, and 85.6-89.4%, respectively. The calibration curve for LAZ was linear in the range of 1.95-250 ng/mL. The inter-day and intra-day accuracy and precision values for LLOQ, low, medium, high and very high concentration QC samples were within ±15%. LAZ was tested under different storage conditions, for short-term bench-top stability (1h and 3h at 25°C), long-term stability (1 month at -80°C), freeze-thaw cycle stability (1 cycle and 3 cycles) and stability of processed samples in auto-sampler (24h at 10°C) with stability values within ±15% range of nominal concentrations. The validated UPLC-MS/MS method was further applied to a pharmacokinetic study in rats after a single intravenous dose of LAZ at 5 mg/kg.

Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains.

Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR.

[The Effect of the Antioxidant Drug U-74389G on Magnesium Levels During Hypoxia-Reoxygenation Injury in Rats].

OBJECTIVE: The aim of this experimental study was to examine the effect of the antioxidant drug U-74389G in a rat model of hypoxia-reoxygenation using the previously established protocol. Effects of treatments were evaluated by magnesium (Mg2+) levels in blood. METHODS: Non-randomized controlled study was performed. Mg2+ levels were determined in 60 min (groups A and C) and 120 min (groups B and D) after starting the reoxygenation. Groups A and B received no drugs, whereas rats from groups C and D were administered with U-74389G. RESULTS: 40 rats 16-18 weeks old of a mean weight of 2312 g were employed in the study. It is demonstrated that U-74389G administration did not alter the Mg2+ levels (decrease in Mg2+ concentration was 0.28±2.75%; p=0.917). Reoxygenation non-significantly increased the Mg2+ levels by 4.27±2.66% (p=0.107). Together, the U-74389G administration and reoxygenation non-significantly increased the Mg2+ levels by 0.36±1.64% (p=0.823). CONCLUSION: U-74389G administration, alone or in concert with reoxygenation did not significantly affect Mg2+ level in blood after experimental hypoxia in rats.

Effects of U-74389G (21-Lazaroid) and Ascorbic Acid on Liver Recovery After Acute Ischemia and Reperfusion in Rats.

BACKGROUND/AIM: The free radical-scavenging effects of the lazaroid U-74389G have been shown in several experimental models to protect the liver from ischemia/reperfusion (I/R), however, the mechanism of cytoprotection is not fully understood. Similar findings were observed when ascorbic acid was administered. This study investigates the effects of infusion of lazaroid U-74389G and ascorbic acid on cytokines and liver structure in a liver I/R rat model. MATERIALS AND METHODS: Sixty male Wistars rats, weighting 220-290 g, were used in the study. Six experimental groups were formed: Group 1 (control group): ischemia for 30 min and reperfusion for 60 min; group 2 (control group): ischemia for 30 min and reperfusion for 120 min; group 3: ischemia for 30 min, intraportal injection of ascorbic acid, and reperfusion for 60 min; group 4: ischemia for 30 min, ascorbic acid administration, and reperfusion for 120 min; group 5: ischemia for 30 min, U-74389G administration, and reperfusion for 60 min; and group 6: ischemia for 30 min, U-74389G administration, and reperfusion for 120 min. Tissue and blood sampling took place upon completion of each model's reperfusion. U-74389G was administered at 10 mg/kg animal body weight and ascorbic acid at 100 mg/kg. Anesthesia was induced with ketamine and xylazine. Surgery was performed through a midline laparotomy. The portal vein and the common hepatic artery were isolated and prepared for occlusion. Blood samples and wedge liver biopsies were taken to measure levels of liver enzymes, cytokines and for microscopic analysis upon completion of reperfusion once for each model. RESULTS: Histopathological evaluation revealed a statistically significant reduction in the degree of necrosis of liver tissue in the treated groups compared to the control groups 1 and 2 [groups 3, 5 (p=0.010) and 4, 6 (p<0.0005)]. On the other hand, tissue malondialdehyde levels (MDA) were statistically significantly increased only between control group 2 and groups 4, 6 (p<0.0005). There was no statistically significant difference in tumor necrosis factor-α between groups. As for liver enzymes, only alkaline phosphatase (ALP) and gamma-glutamyl transferase (gGT) were statistically significantly reduced in treated groups 3 and 5 (ALP: p=0.027, and gGT: p=0.002) and 4 and 6 (ALP: p=0.004, and gGT: p=0.015) compared to control groups 1 and 2. CONCLUSION: Based on histological data and the reduction of some of the liver enzymes, in spite of a rise of malondialdehyde, in this rat model, administration of U-74389G in liver ischemia/reperfusion (I/R) injury has potential in attenuating liver damage.

Neuroprotective Role of Lazaroids Against Aluminium Chloride Poisoning.

Aluminium (Al) is neurotoxic primarily because of its interference with biological enzymes in key mechanisms of metabolic pathways. Mitochondria being a major site of reactive oxygen species (ROS) production, it seems that the oxidative damage to mitochondrial proteins may underlie the pathogenesis of Al induced neurodegeneration. The present study investigates the effectiveness of the anti-oxidant property of lazaroids (U-74500A), a known lipid peroxidation inhibitor as neuroprotective agent against Al induced neurotoxicity. Al chloride was administered orally at a dose level of 100 mg/kg body wt/day in water and U-74500A was administered at a dose of 0.25 mg/kg body wt i.p. in citrate buffer for a period of 8 weeks on alternate days. Following Al exposure there was a significant increase in lipid peroxidation (LPO), ROS levels and reduction in the activity of mitochondrial complexes in all the three regions of rat brain, i.e., cerebral cortex, mid brain, and cerebellum. This decrease in the activities of electron transport complexes in turn affected the ATP synthesis and ATP levels adversely in the mitochondria. These alterations were also depicted in the histology which shows signs of hypoxia, paucity of neurons in cortical region and loosening of fibers in the white matter. U-74500A co-administration was able to restore alterations in the LPO, ROS levels as well as all the three mitochondrial complexes and caspase expression. Therefore, it is suggested that 21-aminosteroids (lazaroids), by attenuating LPO and mitochondrial dysfunction, holds a promise as an agent that can potentially reduce Al-induced adverse effects in brain.