Cell and Tissue Journal

Abstract Introduction: Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer in polyvinyl chloride (PVC) products, including medical devices such as syringes, IV tubing, blood bags, respiratory circuits, and dialysis equipment. Due to its weak physical bonding to the PVC matrix and lack of covalent interactions, DEHP can leach into biological fluids upon contact. This becomes particularly concerning during prolonged clinical exposure, as it allows the compound to enter the bloodstream and potentially affect sensitive tissues, including bone marrow. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent progenitors with the ability to differentiate into osteoblasts, playing a vital role in bone remodeling and regeneration. Given their sensitivity to environmental toxins, DEHP exposure represents a significant risk to BMSC viability and osteogenic function. Evidence suggests that DEHP disrupts osteogenesis by inducing oxidative stress and downregulating essential genes involved in matrix formation and mineralization. Curcumin (Cur), a bioactive polyphenol extracted from the rhizome of Curcuma longa, possesses strong antioxidant, anti-inflammatory, and cytoprotective properties. It is nontoxic, affordable, and widely available, making it a promising candidate for counteracting xenobiotic-induced oxidative damage. This study explores whether Curcumin can mitigate DEHP’s deleterious effects on BMSCs during osteogenic differentiation.
Aim: We hypothesize that co-treatment with Cur will ameliorate DEHP-mediated disruption in cell viability, antioxidant capacity, and osteogenic marker expression.
Materials and Methods: BMSCs were isolated from adult male Wistar rats under sterile conditions and cultured to passage three. Cells were divided into four experimental groups and exposed for 21 days to the following treatments: (1) control, (2) DEHP (100 μM), (3) Curcumin (0.1 μM), and (4) DEHP + Curcumin. The cells were cultured in osteogenic differentiation medium throughout the treatment period. Cell viability was assessed using the tetrazolium-based MTT assay. Osteogenic differentiation was evaluated via Alizarin Red staining for mineral deposition, calcium quantification, and alkaline phosphatase (ALP) activity measurement. Oxidative stress was assessed by quantifying intracellular malondialdehyde (MDA) levels, a lipid peroxidation marker. Antioxidant enzyme activity for catalase (CAT) and superoxide dismutase (SOD) was measured spectrophotometrically. Total antioxidant capacity (TAC) was evaluated using commercial kits. For molecular analysis, total RNA was extracted from cells and reverse-transcribed into complementary DNA (cDNA). Semi-quantitative PCR was performed to measure expression levels of osteogenic differentiation-related genes: Smad1, Bmp2, Bmp7, Runx2, Alp, Col-1A1, and Osteocalcin (Oc). Gapdh served as the internal control. Data were statistically analyzed using ANOVA with Tukey’s post-hoc test. A threshold of p < 0.05 was considered statistically significant.
Results DEHP treatment caused a marked reduction in BMSC viability (p < 0.0001), confirming its cytotoxicity. Cells co-treated with Curcumin showed a significant restoration in viability (p < 0.01), indicating Cur’s protective effects. Alizarin Red staining revealed diminished extracellular matrix mineralization in DEHP-treated cells, with corresponding reductions in calcium content and ALP activity. Notably, Curcumin co-treatment restored all markers to levels comparable to those seen in control cells. ALP, an early osteogenic marker, showed complete recovery, underlining Cur’s efficacy in preserving bone-forming potential. Gene expression analysis demonstrated that DEHP downregulated osteogenic genes (Smad1, Bmp2, Bmp7, Runx2, Col-1A1, Oc). Co-treatment with Curcumin significantly reversed this suppression, elevating transcript levels to near control values. These results suggest a transcriptional rescue linked to improved redox homeostasis. Oxidative stress measurements indicated that DEHP increased MDA levels while suppressing CAT and SOD activity, as well as total antioxidant capacity (p < 0.0001). Curcumin treatment effectively reduced MDA concentrations (p < 0.05), and boosted CAT and SOD activity (p < 0.01). TAC was significantly elevated in Curcumin-treated groups (p < 0.0001), indicating improved redox balance and defense against oxidative damage.
Conclusion: This study provides strong evidence that DEHP impairs BMSC viability and osteogenic differentiation primarily through oxidative stress mechanisms. Curcumin co-treatment mitigates DEHP-induced cellular damage, restores osteogenic function, and enhances antioxidant defenses. These findings highlight Curcumin’s potential as a therapeutic agent to counteract phthalate toxicity in clinical contexts involving prolonged exposure to DEHP-containing materials.

Abstract Aim: Di-2-ethylhexyl phthalate (DEHP) is used as plasticizer to produce flexible polyvinyl chloride (PVC) which is used in food and medical industries. Due to temperature change and contact with biological fluids and other liquids, DEHP leaches out from PVC. Humans get exposed to DEHP in different way including food consumption and medical utilities such as blood bags, blood transfusion tubes, syringes. At 2002, 120000 tons of DEHP has been produced in United States of America and this production was raised up to 230000 tons at 2006. In Iran, only in one of the industries called as Farabi petrochemical industries, 55000 tons of phthalate per year is produced. It has been reported that the DEHP concentration in human blood might reach to 52 to 55 µg/ml when blood is stored in polyvinyl chloride bags for two weeks.  Previously, the effect of different concentration of DEHP on viability, proliferation and differentiation of rat bone marrow mesenchymal stem cells was investigated. In the present study, effect of 100 µM (39 µg/ml) of DEHP on the expression of genes related to osteogenic differentiation of rat bone marrow mesenchymal stem cells was investigated.
Material and methods: In this experimental study, rat bone marrow mesenchymal stem cells were extracted from Wistar rats and after 3^rd passage the cells cultured was performed in osteogenic media in presence of 100 µM of DEHP for 21 days. The viability of the cells was studied using 3-[4, 5-dimethylthiazol-2yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. In addition, the osteogenic differentiation of the rat bone marrow mesenchymal stem cells was investigated using quantitative alizarin red test and calcium concentration determination. In addition, alkaline phosphatase enzyme activity as a marker of osteogenic differentiation was measured.  Expression of the osteogenic related genes (osteonectin, SMAD1، BMP2، BMP7 and RUNX2) were studied using reverse transcriptase-PCR. Data was analyzed and the minimum level of significant was considered as p<0.05.
Results: Data analysis revealed, rat bone marrow mesenchymal stem cells viability reduced significantly (p<0.05) following treatment with 100 µM of DEHP when compared to control one. Also, we observed a significant (p<0.05) reduction in matrix production based on significant reduction in alizarin red concentration as well as calcium content and alkaline phosphatase enzyme activity. In addition, a significant (p<0.05) reduction in total protein also was observed in the samples extracted from BMSCs differentiated to osteoblasts in presence of DEHP. Meanwhile, a significant (p<0.05) down regulation of osteogenic related genes was confirmed while no change (p>0.05) was observed in the expression of GAPDH.
Conclusion: based on this study, long term exposure to DEHP caused reduction in matrix production which strongly showed the osteogenic differentiation of rat bone marrow mesenchymal stem cells is affected by low concentration of this environmental pollutant. As it was observed, the concentration used in this study was lower than the concentration of this pollutant in blood bags. Therefore, if a patient gets exposed to the biological or non-biological fluids in a treatment procedure, large amount of DEHP would enter the blood circulation system. As DEHP is used in production of food containers and medical utilities such as blood tubing, blood bags, dialysis tubes in dialysis machines, we strongly suggest a legal restriction to be forced on the industrial which using this chemical as plasticizer.

Abstract Aim: Nowadays, much attention is paid to the effects of natural factors on physiological and pathological processes in human body. In this regard, lack of oxygen or hypoxia is one of the crucial biological factors involved in various physiological processes such as wound healing and pathological processes such as cancer. Moreover, it is very important to find natural compounds affecting the characteristics and functions of cells. Thymoquinone (TQ) is a natural compound derived from certain plants such as Nigella Sativa. It has many biopharmacological effects, including anti-bacterial, anti-oxidant, anti-inflammatory, anti-diabetic, anti-aging, anti-cancer, etc. Given the biopharmacological properties of TQ and the importance of hypoxia as an important factor affecting physiological and pathological processes, this study was designed to investigate the effect of TQ under cobalt (II) chloride-mediated hypoxia on breast cancer and wound healing by evaluating the expression of SOX2, CDK4, c-MET, and DNMT1 genes in a breast cancer cell line (MCF7) and a normal fibroblastic cell line (HDF) that treated with these compounds.
Materials and Methods: In the present study, after the cultivation of MCF7 and HDF cell lines, each of the cells were divided into two groups. The treatment group was treated simultaneously with 500 ng/ml of TQ and 100 μM of cobalt (II) chloride for 24 h and the control group was only treated with cobalt (II) chloride. After incubation time, total RNA extraction, DNase I treatment, and cDNA synthesis were carried out and finally, the expression of target genes was examined by real-time PCR assay. In this study, relative threshold method was used to determine the amount of gene expression changes, and SPSS software and Student's t-test statistical method were used to find the significance of gene expression changes in the treated groups compared to the controls.
Results: The results showed that simultaneous treatment of MCF7 cells with TQ and cobalt (II) chloride significantly (P < 0.05) reduced the expression of CDK4, c-MET, and DNMT1 genes at about 4.35-, 1.89-, and 2.08-fold, respectively, compared to the control group. However, the treatment of MCF7 cells caused a limited increase in the expression of SOX2 at about 1.14-fold, which was not significant according to the significance level of ≥ 1.5. Moreover, simultaneous treatment of HDF cells with TQ and cobalt (II) chloride significantly increased c-MET gene expression by about 1.86-fold. In addition, the treatment of HDF cells caused a slight increase in the expression of CDK4 at about 1.26-fold, which was not significant according to the significance level of ≥ 1.5. Also, the expression of SOX2 and DNMT1 genes has decreased at about 1.28- and 1.32-fold in the treatment group compared to the control group, which were as not significant according to the significance level of  ≥ 1.5.
Conclusion: Overall, it can be concluded that TQ under cobalt (II) chloride-mediated hypoxia may inhibit breast cancer by inhibiting the expression of genes involved in proliferation and migration. In addition, due to the important role of fibroblasts in the wound healing process, TQ may help wound healing under hypoxic conditions by increasing the migration potential of fibroblast cells.

Abstract Aim: Thepurpose of this study is the investigation of the effect of aqueous and ehanolic plant extracts including neem, clove, thyme and lavender on the increase storage life via decrease of reactive oxygen and the activity of antioxidant enzymes.
Material and methods: In this study, it was extracted of the neem, clove, thyme and lavender with aqueous and ehanolic solution, then the orange fruits was treated with 2×1000, 4×1000 and 6×1000 concentration of plant extract and chitosan and vax. The treated fruits were stored in the storage with 7C and 80-90% humidity. The enzyme activity and the related genes expression was evaluated per twenty days to 100 days in the orange fruits. In the other section of study, it was done the panel test.
Results: The results showed the activity of catalase was affected with plant extracts. The treated orange fruits with 6×1000 concentration of ethanolic lavender extract showed the least catalase activity, with 2.13; the least peroxidase activity was observed in the fruits treated with aqueous lavender extract with 6×1000 concentration. The ethanolic and aqueous of lavender extract affected on the catalase and peroxidase gene expression with 5.28 and 6.66 respectively.
Conclusion: the results of this study showed the extracts of neem, clove, thyme and lavender have highly effect on the plant physiology and they decreased the enzyme activity and the genes expression.

Abstract Aim: Evaluation effects of ZnO nanoparticles on peroxidase activity, gene expression of peroxidase enzyme, proline content and expression of proline-5-carboxylate synthetase (P5CS) gene in L. draba seedlings in compared to the effects of its balk form.
Material and Methods: L. draba seedlings were grown in the presence of different concentrations of these particles for 7 days in fully-random designs with three repetitions; thereafter the mentioned parameters were measured.
 Results: While the peroxidase activity was significantly evaluated in the presence of nano and balk particle in the concentration more than 100 and 50 mg/L respectively, in them, the gene expression level of peroxidase was the same as the control. On the other hand, in the treated seedlings, proline content was significantly increased in accordance to the rising nanoparticle doses in media, while, no significant difference was seen in the P5CS gene expression in different treatments rather than control sample. In the balk treated seedlings, content of this amino acid were significantly increased up to 100 mg/L concentrations and dramatically decreased in the presence of doses more than 250 mg/L in compared to the control sample. Gene expression level of P5CS was the same as the control in the presence of the balk form up to 500 mg/L and significantly decreased at the highest concentration.
Conclusion: Based on the results, it seems that nanoparticle has fewer effects rather than the balk form on the mentioned parameters, which may be due to release less Zn ion in this condition.

Abstract Aim: The aim of this study was to evaluate the effect of walnut green skin extract on blue mold apple, peroxidase activity and catalase activity, as well as the level of gene expression of these two enzymes.
Material and methods: In this study, extracts of green skin of walnut were used to control the apple blue mold caused by Penicillium expansum, in vitro and in vivo. Then, the effect of the aqueous extract of this plant on the activity and also expression of the genes of the two enzymes of peroxidase and catalase were evaluated.
Results: The results of the extract mixed with culture media tests showed the aqueous and methanolic extracts of green walnut skin inhibit with the rate of 86.41 and 75.84 percent of the fungal pathogen were respectively the best treatment in comparison with the control. In vivo test (the 4 ° C), the spot surface, in the treatment of aqueous and alcoholic extracts with a concentration of  6×1000, reduced 94.50 and 81.69%, respectively. In the test for the activity of peroxidase and catalase activity, the walnut skin extract increase the activity of these enzymes at 9th day. The results of enzymes genes expression showed that the expression of peroxidase and catalase genes was 227.66 and 314.08 fold on day 9, in the plant extract+ pathogen treatment, respectively.
Conclusion: The extract of walnut skin had direct fungal effects, and it can was induce the defense genes expression and subsequently increased activity of defense enzymes.
 
 

Abstract Aim: Effects of two nanoparticles were investigated on the expression of STR, DAT and D4H genes in periwinkle.
Material and Methods: For this purpose, concentrations of 0.5, 0.75 and 1 mg per liter of cobalt and zinc oxide nanoparticles were used. Sampling was carried out at 0, 8, 24 and 48 hours after treatment.
Results: Gene expression analysis was performed by the SQ-RT-PCR method. In general, both elicitors influenced gene expression. But, the effects of zinc oxide nanoparticles on the gene expression were more pronounced than cobalt oxide nanoparticles. The highest expression of STR and D4H genes were occurred in 0.5 mg per liter of zinc oxide nanoparticles after 8 hours treatment, while in the case of DAT gene, it occurred in concentration of 1 mg per liter of this nanoparticle. Moreover, Cobalt oxide nanoparticles in most of the studied concentrations and time intervals caused decreases in gene expression.
Conclusion: Both nanoparticles had significant effects on gene expression of vinblastine and vincristine pathways.

Abstract Aim: The aim of this study was to investigate the expression of key genes, 1-deoxy-D-xylulose-5-phosphate reductoisomerase )DXR( and gamma-terpinene synthase (GTS), involve in thymol and carvacrol biosynthesis pathway in Summer savory (Satureja hortensis). This species is one of the important medicinal plants of the Lamiaceae family, and consider as an important source of the mentioned compounds.
Material and Methods: Plants were treated with salicylic acid, methyl jasmonate and UV-B rays. RNAs were extracted from control and treated plants and cDNAs were synthesized. Primers were designed for gene isolation and expression studies. Transcript expression analyses for the DXR and GTS were performed using semi-quantitative RT-PCR method.
Results: A partial segment for DXR and GTS genes was sequenced. The relative gene expression analyses showed differential expression of both genes at transcript level in different tissues (roots, stems, leaves and inflorescence), with higher levels of expression in leaf and inflorescence. The expression of both genes under the effect of abiotic elicitors including salicylic acid, methyl jasmonate and UV-B rays exhibited significant alteration.
Conclusion: Under controlled conditions, using abiotic elicitors such as: salicylic acid, methyl jasmonate and UV-B radiation could elevate the level of gene expression and possibly increase the production of secondary metabolite such as: thymol and carvacrol.

Abstract Aim: The purpose of the present research project was evaluation of the effect of silicon on the severity of peroxidase genes expression and some morphological traits in both resistant and susceptible barley lines under drought stress. Material and Methods: Total soluble protein content, photosynthetic pigments, and total RNA were extracted from the leaves affected by various treatments. Target gene was evaluated by Semi-quantitative RT-PCR analysis using synthesized cDNA. Both the peroxidase enzyme activity by Chance method and proline with Bates method were also evaluated under drought stress treatment. Accordingly, the experiment was analyzed in a factorial test based on the completely randomized design with three treatments of control, drought and silicon-drought (sodium silicate 2 mg / 1 Kg soil) with three replications in a greenhouse. Results: Silicon application caused to increase the amount of total soluble protein and photosynthetic pigments in both lines under drought stress. Semi-quantitative RT-PCR analysis of treatments observed significant differences. Maximum of peroxidase gene expression was observed in the silicon-drought treatment. Antioxidant enzyme activities were at the highest level as shown by gene expression pattern of polyacrylamide gel analysis for treated silicon samples. A further increase was exhibited for proline accumulation caused by silicon application rather than such accumulation in stress treatment compared to control. Conclusion: Silicon reduces oxidative damage induced by reactive oxygen species and with the increase in antioxidant enzyme activities caused to protect plants against environmental stresses.  

Abstract Aim: Our aim in this study was to analyze and quantify mRNA expression of SALL4 in mesencephalon, during different stages of chicken embryogenesis. Material and methods: In this experimental study, incubated Ross fertilized eggs were applied in 37°C-37.5°C in 60-65% humidified atmosphere after beginning of embryogenesis. Mesencephalon part of the brain tissue was collected from the eggs, daily. Total RNA extraction and cDNA synthesis was performed from resected tissues. The synthesized cDNA was used as template for quantitatively analysis of SALL4 mRNA expression by real-time PCR. Results: The Real-time PCR analysis of SALL4 mRNA expression in mesencephalon tissues indicated that the level of gene expression is significantly variable during embryogenesis. While the basal level of SALL4 mRNA expression was detected during the rest of embryogenesis, the maximum copy number of SALL4 mRNA was quantified on 19th day of chicken development. Conclusion: Having analyzed the level of SALL4 mRNA expression in different stages of chicken embryogenesis, we can extrapolate that a probable relationship may be existed between expression of SALL4 in nerve centers of mesencephalon brain and development of optic organs. Keywords: