Evaluation of Beneficial Influence of Local Application of Crocus Pallasii Subsp. Haussknechtii Boiss. Extract on Healing of Full Thickness Excisional Infected Wounds in Diabetic Rats.

OBJECTIVE: To evaluate the wound healing activity of Crocus pallasii subsp. haussknechtii boiss leaves extract on infected wounds in diabetic rats. METHODS: Fifty male diabetic rats were randomized into two sets of 25 animals each. Each group was sub divided into five groups of five animals, each for excisional and incisional wound models, respectively. Induction of diabetes was achieved using 60 mg/kg streptozotocin. In group I, 0.1 mL sterile saline 0.9% solution was added to the wounds with no infection. In group II, the wounds were infected with Methicillin-resistant staphylococcus aureus (MRSA) and only treated with 0.1 mL the sterile saline 0.9% solution. In group III, infected wounds were treated with application of base formulation ointment. In group IV, animals with infected wounds were treated with 0.1 mL topical application of 1 mg/mL methicillin and base formulation ointment. In group V, animals with infected wounds were treated with topical application of 0.1 mL solution of methicillin (1 mg/mL) and with 1g of powder extract of the plant material in ointment. The healing of the wound was assessed based on planimetry, hydroxyproline estimation, microbiological, biomechanical and biochemical studies. RESULTS: Microbiological examination, planimetric, histological and quantitative morphometric studies and determination of hydroxyproline levels showed that there was significant difference between animals in group V compared to other groups (p=0.001). Biomechanical indices in incisional groups showed there was significant difference between animals in group V compared to other groups (p=0.001). CONCLUSION: It was possible to conclude that the ointment of the extract of Crocus pallasii subsp. haussknechtii boiss. leaves have significant wound-healing activity in diabetes.

Glycine supplementation mitigates lead-induced renal injury in mice.

PURPOSE: Lead (Pb) is an environmental pollutant responsible for various organ damages including renal injury. It seems that OS and associated events are crucial mechanisms of lead-induced renal dysfunction. The current study aimed to explore the potential protective effects of glycine against renal injury caused by lead in mice. MATERIALS AND METHODS: Mature male mice (n=32) were allocated into four groups. The following treatment regimens were the control (vehicle-treated); Pb-acetate (20 mg/kg/day, gavage); Pb-acetate + glycine (500 mg/kg/day, IP); and Pb-acetate + glycine (1,000 mg/kg/day, IP). Pb-acetate + glycine was administered for 14 consecutive days, Pb-acetate was given first and then glycine at least 6 hours later. On day 15, the subjects were anesthetized, and samples were collected. Serum biomarkers such as BUN and serum creatinine were monitored along with formation of reactive oxygen species, lipid peroxidation, kidney GSH level, and histopathological changes. RESULTS: Based on the results, BUN and serum creatinine levels significantly increased following exposure to lead. Glycine supplementation (500 and 1,000 mg/kg, IP) decreased BUN and creatinine serum levels (P<0.001). Biomarkers of OS were also reduced in renal tissue following glycine therapy in Pb-exposed mice (P<0.001). Histopathological changes were observed in mice treated with lead as tubular dilation, protein cast, vacuolization, and inflammation. In this regard, glycine inhibited histopathological alterations in kidney caused by lead exposure. CONCLUSION: It was found that glycine treatment significantly mitigated Pb-induced renal injury most likely through alleviating OS and the associated deleterious outcomes on the kidney tissue.

Analysis of effectiveness of Iranian snake antivenom on Viper venom induced effects including analysis of immunologic biomarkers in the Echis carinatus sochureki envenomed victims.

Snakebite is an important toxicologic emergency with the potential of triggering local and systemic inflammation. Antivenom has remained the mainstay of treatment for snakebite envenomation. In this study we sought to investigate the effectiveness of Iranian antivenom in a series of 44 viper envenomed patients through analysis of changes in clinical severity and the levels of inflammatory markers. Clinical envenomation severity assessed by snakebite severity score (SSS) and laboratory exams of the patients were recorded before (baseline visit) and after antivenom therapy. During 12-h antivenom therapy, the median (range) score of SSS significantly decreased from 3.5 (2-10) on admission to 1 (0-5) in the last visit (P < 0.001). Moreover, a significant decrease in prothrombin time and international normalized ratio was found (P = 0.006 and 0.008; respectively). Plasma concentrations of interleukin (IL) 1-ß, IL-6, IL-8, tumor necrosis factor α (TNF-α), complement hemolytic activity (CH50) were also measured in 10 severely Echis carinatus sochureki envenomed victims and 10 age and gender-matched healthy controls. Except IL-8, the baseline levels of IL-1ß, IL-6 and TNF-α in victims were significantly higher than healthy controls (P = 0.005, <0.001 and < 0.001, respectively). Moreover, the baseline level of CH50 was significantly lower in the patients compared to healthy controls (P < 0.001). After 12-h antivenom therapy, the plasma levels of IL-1ß, IL-6 and TNF-α significantly decreased (P = 0.032, 0.006 and 0.003, respectively), the levels of IL-8 remained relatively unchanged and the CH50 significantly increased (P = 0.011). Iranian snake antivenom was effective in treating viper bite envenomation as it reversed clinical venom effects and restored near normal underlying inflammatory status. This study is the first to ascertain and report the effectiveness of this antivenom in human subjects.

Solvent-stir bar microextraction system using pure tris-(2-ethylhexyl) phosphate as supported liquid membrane: A new and efficient design for the extraction of malondialdehyde from biological fluids.

A novel and efficient device of solvent stir-bar microextraction (SSBME) system coupled with GC-FID detection was introduced for the pre-concentration and determination of malondialdehyde (MDA) in different biological matrices. In the proposed device, a piece of porous hollow fiber was located on a magnetic rotor by using a stainless steel-wire (as a mechanical support) and the whole device could stir with the magnetic rotor in sample solution cell. The device provided higher pre-concentration factor and better precision in comparison with conventional SBME due to the reproducible, stable and high contact area between the stirred sample and the hollow fiber. Organic solvent type, donor and acceptor phase pH, temperature, electrolyte concentration, agitation speed, extraction time, and sample volume as the effective factors on the SSBME efficiency, were examined and optimized. Pure tris-(2-ethylhexyl) phosphate (TEHP) was examined for the first time as supported liquid membrane (SLM) for the determination of MDA by SSBME method. In contrast to the conventional SLMs of SBME in the literature, the SLM of TEHP was highly stable in contact with biological fluids and provided the highest extraction efficiency. Under optimized extraction conditions, the method provided satisfactory linearity in the range 1-500 ng mL-1, low LODs (0.3-0.7 ng mL-1), good repeatability and reproducibility (RSD% (n = 5) < 4.5) with the pre-concentration factors higher than 130-fold. To verify the accuracy of the proposed method, the traditional spectrophotometric TBA (2-thiobarbituric acid) test was used as a reference method. Finally, the proposed method was successfully applied for the determination and quantification of MDA in biological fluids.

Effect of Crude Venom of Odonthobuthus doriae Scorpion in Cell Culture using Ion Channel Modulators.

Scorpion venom toxicity is one of the major medical concerns from old years, due to its influence on human activities and health. From many years ago a lot of researches established to examine different aspects of venom toxicity and its effects on different organs. During these years researchers are doing more specific studies on the cytotoxicity of scorpion venom. In Iran, Odonthobuthus doriae, the yellow scorpion is one of the major threats based on its neuro toxicity and severe pathophysiologic effects and researchers tried to find the mechanism of these neuro toxic effects. The previous studies have shown that in isolated organs the yellow scorpion venom is affecting the ion channels. Also some studies showed that this venom has severe cytotoxic effects on the cell lines with many ion channels like nerve cell lines. In this study, the cytotoxic effect of the crude venom of O.doriae on the 1321N1 cell line (cancerous nerve cells) was studied. Primary cell cultured investigated in the presence of different ion channel blockers: Ouabain (1mmol as Na channel blocker), Nifedipin (100 µmol as Ca channel blocker), and TEA (40 mmol as K channel blocker) by MTT method. The result showed that the O.doriae crude venom has cytotoxic effect via Na channels.

Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture.

Nanotechnology is a most promising field for generating new applications in medicine, although, only few nano products are currently in use for medical purposes. A most prominent nanoproduct is nanosilver. Nano-silver has biological properties which are significant for consumer products, food technology, textiles, and medical applications (e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging). For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products. The use of nano-silver is becoming more and more widespread in medicine and related applications, and due to its increasing exposure, toxicological and environmental issues need to be raised. Cytotoxicity induced by silver nanoparticles (AgNPs) and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and they induced intracellular oxidative stress. AgNP reduced ATP content of the cell and caused damage to mitochondria and increased production of reactive oxygen species (ROS) in a dose-dependent manner. Silymarin was known as a hepatoprotective agent that is used in the treatment of hepatic diseases including viral hepatitis, alcoholic liver diseases, Amanita mushroom poisoning, liver cirrhosis, toxic and drug-induced liver diseases. It promotes protein synthesis, helps in regenerating liver tissue, controls inflammation, enhances glucuronidation, and protects against glutathione depletion. Vitamin E is a well-known antioxidant and has hepatoprotective effect in liver diseases. In this study, we investigated the cytotoxic effects of Ag NPs on primary liver cells of mice. Cell viability (cytotoxicity) was examined with MTT assay after primary liver cells of mice exposure to AgNPs at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration- dependent decrease of cell viability (IC50 value = 121.7 ppm or µg/ml). Then the hepatoprotective effect of silymarin and vitamin E were experimented on silver nanoparticle toxicity on mice liver primary cell culture. The results showed that silymarin at 600 µg/ml and vitamin E at 2500 µmol/l have protective effects on silver nanoparticle toxicity on mice liver primary cell culture. Viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles at 121.7 ppm and co-treatment of silymarin, and vitamin E is more than viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles and silymarin or silver nanoparticles and vitamin E.

Effectiveness of a clinical protocol implemented to standardize snakebite management in Iran: initial evaluation.

OBJECTIVE: This study was designed to evaluate the effectiveness of a new protocol implemented to standardize snakebite management in Iran. METHODS: In this study, 27 patients treated according to the new protocol in 2012 (P+) were compared with 22 patients treated according to the previous modality in the year before implementation of the protocol (P-) in Mashhad Medical Toxicology Centre (MTC). Demographic characteristics and treatment details of all patients were recorded prospectively. Envenomation severity of each victim was assessed according to snakebite severity score (SSS). RESULTS: After implementation of the protocol, a smaller percentage of patients received antivenom (AV) therapy (78% vs 95%; P=.079). In spite of no significant difference in baseline severity of envenomation between the 2 groups (SSS [mean±SD], 34.8±18.1 vs 35.5±17.4; P=.801), the P+ group received significantly fewer AV vials (8.4±6.8 vs 12.1±5.6 vials; P=.042) and had a significantly shorter length of hospital stay (2.2±1.5 vs 3.2±1.8 days; P=.027). Moreover, smaller proportion of P+ patients experienced recurrence of venom-induced effects; however, the difference was not significant (18.5% vs 36%; P=.159). The reduction in use of antiallergy treatments to prevent or treat acute hypersensitivity reactions approached statistical significance (41% vs 68%; P=.051). These findings denote a reduction in AV use of approximately 4 vials and a reduction in hospital stay of 1 day for each patient, which translates to approximately $196/patient in healthcare cost savings. CONCLUSIONS: Implementation of a snakebite management protocol at MTC reduced overall antivenom usage, use of antiallergy interventions, and length of hospital stay.

Toxicity Effect of Silver Nanoparticles on Mice Liver Primary Cell Culture and HepG2 Cell Line.

Nano-silver (AgNP) has biological properties which are significant for consumer products, food technology, textiles and medical applications (e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging). For their antibacterial activity, silver nanoparticles are largely used in various commercially available products. Thus, the use of nano-silver is becoming more and more widespread in medicine. In this study we investigated the cytotoxic effects of AgNPs on liver primary cells of mice, as well as the human liver HepG2 cell. Cell viability was examined with MTT assay after HepG2 cells exposure to AgNPs at 1, 2, 3, 4, 5, 7.5, 10 ppm compared to mice primary liver cells at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration-dependent decrease of cell viability in both cells. IC50 value of 2.764 ppm (µg/mL) was calculated in HepG2 cell line and IC50 value of 121.7 ppm (µg/mL) was calculated in primary liver cells of mice. The results of this experiment indicated that silver nanoparticles had cytotoxic effects on HepG2 cell line and primary liver cells of mice. The results illustrated that nano-silver had 44 times stronger inhibitory effect on the growth of cancerous cells (HepG2 cell line) compared to the normal cells (primary liver cells of mice). which might further justify AgNPs as a cytotoxic agents and a potential anticancer candidate which needs further studies in this regard.

Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei.

BACKGROUND AND OBJECTIVES: Pyrococcus woesei is a hyperthermophilic archaea and produces a heat stable polymerase (Pwo polymerase) that has proofreading activity. MATERIALS AND METHODS: In this study, this microorganism was cultured, its DNA was extracted and the pwo gene polymerase was cloned, expressed and purified. The DNA sequence of the cloned gene was verified by sequencing. The pwo polymerase gene consists of 2,328 bps (775 amino acids with about 90 kD molecular weight). Cloning was done by GATEWAY™ Cloning System and for purification of recombinant protein; His6x-Tag was added to the C-terminus of the recombinant protein. RESULTS AND CONCLUSION: We could purify Pwo polymerase enzyme by Ni-NTA resin. PCR assay showed that Pwo polymerase activity is comparable to a commercial Pfu polymerase activity.