Cell and Tissue Journal

Abstract Aim: Oxidative stress is an imbalance between oxidants and antioxidants at the cellular level which leads to infertility in males. For several decades, reactive oxygen species have been known as destructive and damaging agents to cells and tissues. Cells produce small and controlled amounts of ROS to control their physiological activities. Under normal conditions, ROS produced in semen are continuously deactivated by antioxidants in semen, Unsaturated fatty acid chains in sperm plasma membrane are vulnerable to oxidative stress conditions, and thus spermatozoa depend on extracellular antioxidant systems to overcome oxidative stress conditions. Another reason for creating oxidative stress conditions for spermatozoa, in addition to the low level of antioxidants in semen, is excessive production of ROS by spermatozoa with abnormal morphology. so one of the reasons for the creation of oxidative stress in semen is due to the imbalance between ROS production and its inactivation by antioxidants. Exposure to high concentrations of ROS causes disruption of the mitochondrial membrane, plasma membrane, and also chromosome fragmentation, which reduces sperm motility and viability. Increased formation of ROS is associated with decreased sperm motility. There is a possibility that the increase in ROS production will ultimately cause a decrease in the phosphorylation of axonemic proteins and sperm motility. This condition leads to a decrease in fluidity of the membrane, which in turn is necessary for sperm-oocyte fusion. The use of antioxidants such as silymarin can prevent the effects of oxidative stress.Milk thistle is the most well-known English common name for this species and other names include Holy thistle, Mary thistle, St. Mary’s thistle, Marian thistle, Lady’s thistle, Christ’s crown, Venus thistle, Heal thistle, Variegated thistle, Pig leaves, Royal thistle, Snake milk, Sow thistle, and Wild artichoke. The milk thistle medicinal plant is widely used in the traditional medicine of China and most European countries in the treatment of liver and biliary disorders. The seed extract of this medicinal plant, which is known as silymarin, protects the liver against a variety of poisonings. Silymarin contains a collection of flavonoids and other compounds with antioxidant, anti-inflammatory and cellular glutathione-increasing properties. Among the various flavonoid compounds found in the Silybum marianum, are silybin, silychristian and silydianin, which are collectively called silymarin. In many cases, the antioxidant properties of Silymarin are considered to be responsible for its protective actions. Oxidative stress-induced apoptosis in spermatozoa may lead to male infertility. Environmental pollutants and heavy metals cause harmful effects on the reproductive system and sperm parameters through the induction of oxidative stress. Silymarin, as a potent antioxidant, is able to inhibit oxidative stress. Silymarin with its antioxidant properties can have reduced the effects of compounds such as; aluminum, cadmium, lithium, nickel, benzopyrene, doxycycline, tetracarbon chloride, nicotine, methotrexate, arsenic and lead acetate on the sperm. And improve the damaged sperm parameters resulting from the mentioned compounds. Silymarin is also effective in reducing the harmful effects of varicocele and radiation therapy on sperm parameters.
 
 
 

Abstract Aim: Tissue engineering refers to methods that are based on the use of scaffolds, cells and biologically active molecules to produce tissues with specific functions. The purpose of tissue engineering is to build structures that can regenerate, maintain and improve damaged tissue or the whole organ. Today, by using tissue engineering methods, various natural and synthetic scaffolds have been designed that can be used for nerve grafts. The physical, chemical and biological properties of the scaffold must be similar to the extracellular matrix of the body in order to avoid adhesion, growth and support the differentiation of cells. An ideal neural scaffold should have biodegradability, biocompatibility and proper tensile strength. Recently, the use of polycaprolactan as a suitable biodegradable material has been evaluated in many fields of tissue engineering. Antioxidants are among the substances, which seem to be able to prevent neuronal death by reducing the amount of ROS. Flavonoids include many compounds that have various biological effects in the body. Silymarin (Silybum marianum) is a flavonoid that has many effects, including anti-cancer effects and antioxidant properties. Tragacanth is a known natural polymer that has excellent biological properties such as biodegradability, biocompatibility, antibacterial and wound healing ability. It is obtained from the stems and branches of the Asian species tragacanth. It has outstanding structural stability against heat and acidity. The aim of this study is to produce polycaprolactan/ tragacanth /silymarin nanoscaffolds and to investigate the viability of pc12 cells on the scaffold under oxidative stress. Considering that silymarin has antioxidant properties, the use of polycaprolactan/ tragacanth /silymarin nanoscaffolds can prevent neuropathy of nerve cells.
Material and Methods: Scaffolds used in this research were prepared using the electrophoretic method. For this purpose, an electrospinning machine was used, which is equipped with a rotary collector with a thickness of 70 mm and a width of 50 mm. In order to prepare a polycaprolactan/ tragacanth nanoscaffold and load silymarin on it, a 7% polycaprolactan solution (dissolved in acetic acid), 0.7% by weight tragacanth solution (dissolved in acetic acid) and 0.9% by weight silymarin solution were mixed by a magnetic stirrer for 20 minutes, and in order to make the solution uniform, sodium didecyl sulfate (SDS) with a concentration 1 percent by weight of the solvent was added to the solution and the suspension was homogenized for 20 minutes with an ultrasonic device, then the scaffold was prepared by an electrospinning device. . The nanofibers were collected in a period of 6 hours, the sample collection speed was 1 ml per hour, and the nanofiber samples were collected by rotating at 250 rpm. The distance between the injection needle and the scaffold is 12 cm and this process is done at a voltage of 15 kV. The morphology of the scaffold was evaluated by scanning electron microscope (SEM) and the chemical structure of the scaffold was evaluated by FTIR spectroscopy. To investigate the antioxidant properties of the scaffold, glucose 80 mg/L and H2O2, 150 macro L were used.
Results: Examining the morphology and chemical structure of the scaffold showed the proper porosity of the polycaprolactan/ tragacanth scaffold and the successful loading of silymarin on the scaffold. Evaluation of the oxidant properties of the scaffold after 24 hours of PC12 cell culture on it showed the increase in cell viability on the scaffold and the appropriate antioxidant properties of the scaffold.
Conclusion: The results of this research showed that the enrichment of polycaprolactan/ tragacanth scaffold with silymarin increased the proliferation and survival of PC12 cells under oxidative stress. Therefore, this scaffold can be a suitable candidate for tissue engineering in oxidative stress.

Abstract Aim: Cell therapy using mesenchymal stem cells (MSCs) can be a promising tool in regenerative medicine. One of the richest sources of mesenchymal stem cells is fetal bone marrow. Silymarin has strong antioxidant and anti-inflammatory activities with a positive effect on the proliferation of some cells as well as anti-osteoporosis properties. This study aimed to show the effect of silymarin on the differentiation of mesenchymal stem cells derived from the bone marrow of sheep embryos into the osteogenic line.
Materials and Methods: Mesenchymal stem cells were isolated from the bone marrow of sheep embryos. MTT test was performed to investigate the cytotoxicity of silymarin on cells at different concentrations for 24 and 72 hours. Then, cells in one of 8 groups 1: negative control; 2: treated with 10 μmol/liter silymarin in the usual environment, 3: treated with 20 μmol/liter silymarin in the usual environment, 4: treated with 100 μmol/liter estradiol in the usual environment, 5: positive control, 6: treatment treated with 10 μmol/liter silymarin in the differentiation medium, 7: treated with 20 μmol/liter silymarin in the differentiation medium, 8: treated with 100 μmol/liter in the differentiation medium, were cultured for 21 days. To determine the osteogenic differentiation of cells, the deposition of hydroxyapatite ions was examined using alizarin staining, and also, the amount of ALP enzyme secretion was also measured in the studied groups. Results: Comparing the average optical absorption of cells at different concentrations between 24 and 72 hours after treatment showed that the average optical absorption of cells at zero concentration of silymarin after 72 hours of treatment decreased in comparison with those treated for 24 hours (P<0.05), but no significant difference was observed in other concentrations (P>0.05). Examining the level of ALP enzyme secretion, 21 days after treatment with silymarin in the studied groups showed that the highest level of enzyme secretion was in group 8 (P≤0.05). The lowest amount of enzyme secretion was observed in group 1 (negative control) and then in group 2 and group 3 respectively (P<0.05). No significant difference was observed between groups 4, 5, and 6 (P>0.05). Based on the alizarin red staining results, calcium ions deposition was observed in all the groups related to the differentiation medium, which increased in groups 8, 7, 6, and 5, respectively. In the groups cultured in the usual environment, there was no calcification in group 1 and the amount of calcification increased in groups 2, 3, and 4, respectively. In total, the amount of calcification in the differentiation environment groups was higher in comparison with the usual environment.
Conclusion: During this study, Silymarin had no toxic effect on the mesenchymal stem cells derived from the bone marrow of sheep embryos in the studied concentrations after 24 and 72 hours of treatment. It increased the differentiation of the cells into the osteogenic lineage in a concentration-dependent manner. Therefore, it seems that with further studies and identification of the molecular pathways of silymarin's effect, it can be used in cell therapy in order to repair bone lesions.

Abstract Aim: In order to study the efficiency of the tuber specific promoter (Patatin1), the expression of the HBsAg antigen of the hepatitis B vaccine was evaluated using a transient expression method (AgroInfiltration) in the tubers and leaves of potato plants.
Material and Methods: A tuber specific promoter (Pat1) was isolated from the potato plant using a specific primer pairs by Polymerase Chain Reaction (PCR). It was cloned in the upstream of a synthetic optimized codon of the HBsAg gene in the binary plant vector pBI121. In order to compare the tissue specificity of Pat1, the HBsAg gene also was used under the control of a consultative CaMV35S promoter. The genetic constructs were transferred to the tubers and leaves of potato plants using the Agroinfiltration method. An HBsAg antigen content was measured using ELISA method.
Results: The level of HBsAg antigens in the leaves and tubers of potato plants indicated that Pat1 promoter specifically induced HBsAg high expression in the tuber tissues. Very low expression by the Pat1 promoter in the leaf tissues has been also reported and can be partly dependent on the presence of inducing factors in the inoculation media. However, the expression of HBsAg antigen occurs in both leaf and tuber tissues under the consultative promoter CaMV35S.  The results showed that the codon optimized HBsAg gene for potato plant was expressed properly in plant tissues.
Conclusion:  findings indicate that potato tuber specific promoter Pat1 can be used effectively to express the synthetic optimized HBsAg antigen in the transgenic potato plants.
 
 

Abstract Aim: The aim of this study was to evaluate the effect of silymarin and lithium chloride on DNA integrity and nucleus of ram sperm.
Material and Methods: In this study, Farahani's ram testes were obtained from Arak slaughterhouse immediately after ram daily slaughter and transferred to the research laboratory. A few incisions were made in the epididymis, and spermatozoa were  then washed into a sterile falcon tube by Ham's F10 medium. collected spermatozoa of ram were divided into four groups: 1. Sperm at 0 hour, 2. Sperm incubated for180 minutes (control), 3. Sperm treated with lithium chloride for 180 minutes and 4. Sperm treated with silymarin + lithium chloride for 180 minutes. DNA integrity and DNA fragmentation were investigated by acridine orange staining sperm chromatin expersion (SCD) test respectively. Morphological feature of apoptosis in sperm nucleus was assessed using Diff-Quick staining. Data were analyzed using one-way analysis of variance test (ANOVA) followed by Turkey's test .
Results: The percentage of DNA fragment and apoptosis were significantly increased in lithium chloride-treated group compared to the control. In silymarin+ lithium chloride group, Silymarin could signicantly compensate these effect compared to the lithium choloride group.
Conclusion: Silymarin as a potent antioxidant could  prevent toxic effect of  lithium  on DNA  fragmentation and apoptosis in sperm nucleus.
 

Abstract Aim: This study was performed to investigate if silymarin can prevent the adverse effects of sodium arsenite on ram sperm DNA and nuclear integrity.
Material and Methods: Epididymal sperm obtained from Farahani’s ram (Ovis aries) was swim up and divided into five groups: 1. Sperm at 0 hour, 2. sperm at 180 minutes (control), 3. sperm treated with sodium arsenite (10μM) for 180 minutes, 4. sperm treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 minutes and 5. sperm treated with silymarin (20 μM) for 180 minutes. Ram´s sperm DNA integrity was assessed by SCD (Sperm Chromatine Dispersion) test to study DNA fragmentation and Acridine orange staining was used to estimate DNA denaturation (double-strand DNA versus single-strand DNA). To evaluate sperm nuclear integrity, Diff-quick staining was used and sperm nuclear diameter was measured.
Results: DNA fragmentation percent and nuclear diameter of the spermatozoa were significantly increased and decreased, respectively, in sodium arsenite group compared to the control samples. While this toxicant had no effect on sperm DNA denaturation. In silymarin + sodium arsenite group, silymarin was able to significantly ameliorate the adverse effects of sodium arsenite on these sperm parameters compared to sodium arsenite group.
Conclusion: Silymarin as a potent antioxidant could compensate the adverse effects of sodium arsenite on DNA fragmentation and nuclear diameter of ram sperm.