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: Turmeric contains a high amount of curcumin (Cur), which exhibits strong antioxidant activity. Cur binds to antioxidant response elements for gene, thus suppressing reactive oxygen species (ROS). Reactive oxygen species are short-lived, highly electrophilic molecules. When intracellular antioxidants are reduced or ROS accumulate excessively, it brings about the imbalance in the redox state which leading to oxidative stress. DEHP consists of a pair of eight-carbon esters linked to a benzene-dicarboxylic acid ring with chemical formula of C24H38O4. DEHP is used as a plasticizer in many polyvinyl chloride products, especially in medical devices such as intravenous bags and tubing, umbilical artery catheters, blood bags, infusion tubing, enteral nutrition feeding bags, nasogastric tubes and dialysis bags. Due to non-covalent binding to the plastic products, DEHP leaches into biological fluids during medical treatment causing contamination. There is ample evidence that DEHP induces oxidative stress and causes apoptosis in cells. Therefore, the study aimed to investigate the effects of Cur on DEHP-induced oxidative stress in bone marrow mesenchymal stem cells (BMSCs), as DEHP has been found to induce oxidative stress in BMSCs.
 Materialand Methods: Rat BMSCs was extracted and after the 3rd passage, their cell viability and proliferation were examined in the presence of various concentrations (0.05,0.1, 0.25, 1, 2.5 and 5 μM) of Cur for a period of 4 days. Subsequently, a concentration of 0.1 μM Cur was chosen for further assessment of viability and proliferation abilities (measured by population doubling number (PDN)), total protein, malondialdehyde (MDA), total antioxidant capacity (TAC), catalase and superoxide dismutase (SOD) activity as well as the expression of NFkB and Nrf2 both individually and in conjunction with 100 or 500 μM of DEHP (chosen based on previous study) for durations of 4 and8 days. The data underwent statistical analysis with P<.05 considered as the minimum level of significance.
 Results: Concentrations of 0.05, 0.1, and 0.25 μM of Cur were found to have no effect on the viability (P<0.05) of the BMSCs but significantly increased proliferation ability (P<0.0001). Conversely, concentrations of 1, 2.5, and 5 μM of Cur significantly reduced viability (P<0.0001). The concentration of 0.1 μM was chosen for further analysis based on viability tests and PDN analysis. This selected concentration notably increased BMSCs proliferation after 4 days, while higher concentrations (1, 2.5, and 5 μM) reduced both viability and proliferation. Furthermore, this concentration counteracted the effects of DEHP at both day 4 and day 8 by reducing MDA levels and increasing TAC levels as well as catalase and SOD activity - particularly at day 8 compared to the DEHP-treated groups. Additionally, Cur was found to reduce NFkB expression while increasing Nrf2 expression in BMSCs.
Conclusion: Curcumin mitigated oxidative stress induced by DEHP through the Nrf/NFkB pathway. Moreover, it enhanced the cell viability and proliferation capacity of BMSCs in the presence of DEHP. Therefore, patients undergoing hemodialysis and blood transfusion, who may be exposed to DEHP from medical devices like tubes and other plastic products, should consider consuming turmeric to counteract the oxidative impact of this chemical.

Abstract Aim: Organotypic cultures of spinal cord slices from mammalian neonatal and fetal animals are powerful tools for studies of spinal cord injury, neuronal degeneration, and cell death but also motor neuron regeneration. Models in which adult slices are used would be very useful. However, adult spinal cord slices are notoriously difficult to maintain in culture and rapidly deteriorate in vitro. Degeneration of motor neurons in the spinal cord is a critical phenomenon in spinal cord injuries and certain neurodegenerative diseases such as amyotrophic lateral sclerosis, a neurodegenerative disorder in which motor neurons in the spinal cord and motor cortex are lost. A variety of mechanisms have been proposed as having a role in neuronal apoptosis during spinal cord injury. Oxidative stress has been reported as one of the mechanisms involved in the apoptosis of motor neurons in spinal cord injuries and neurodegenerative diseases. This study was conducted to determine whether quercetin, as a potent antioxidant, can delay apoptosis in motor neurons of cultured spinal cords by reducing oxidative stress.
 Material and methods: The thoracic regions of the spinal cord from adult NMRI mice were sliced using a tissue chopper and divided into three groups: 1) 0-hour, 2) control group, and 3) group treated with quercetin (100 µM). Spinal cord slices in the 2 and 3 groups were incubated for 6 hours at 37°C in a Co₂ incubator. The viability of the spinal cord slices was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The morphological features of apoptosis and the number of motor neurons were examined using Hoechst and propidium iodide staining. The malondialdehyde was measured to determine lipid peroxidation while the FRAP (ferric reducing antioxidant power) was assessed to evaluate total antioxidant capacity in fresh and cultured spinal cord slices. Results were expressed as mean±SD. One-way analysis of variance (ANOVA) followed by Tucky’s test was used to assess the statistical significances of the data. In all cases, a statistical probability of p<0.05 was considered significant.
Results: After 6 hours (control group) in culture, the viability of the spinal cord slices, the neuron diameter, and the number of healthy motor neurons significantly decreased compared to the 0-hour group. Also, motor neurons showed the morphological features of apoptosis including cell shrinkage, nuclear and chromatin condensation in the control group. A significant increase in the amount of malondialdehyde and a significant decrease in the total antioxidant capacity was also observed compared with the 0-hour group. After 6 hours, quercetin not only increased the viability and the number of healthy motor neurons in the cultured slices but also reduced the morphological features of apoptosis in the motor neurons compared with the control group. In addition, quercetin significantly reduced the amount of malondialdehyde and increased the total antioxidant power in slices cultured for 6 hours.
Conclusion: Oxidative stress might be considered as one of the mechanisms involved in the apoptosis of motor neurons in cultured spinal cord slices and quercetin, as a potent antioxidant, was able to increase the viability of the cultured spinal cord slices and delay the morphological features of apoptosis in the motor neurons through reducing lipid peroxidation and increasing the total antioxidant capacity.

Abstract Aim: The purpose of the current study is to investigate the cytotoxic and oxidative effects of Salvia officinalis hydroalcoholic extract on cancer A549 cells.
Material and Methods: The cells were seeded in plates to investigate concentrations of Salvia officinalis extract and MTT measurement was done to determine IC50 concentration and cell viability on days 1, 3, 5, and 7. Moreover, analyses of superoxide dismutase and catalase enzymes involved in oxidative stress pathway, and AO/EB staining for a qualitative investigation of cell lines has been conducted in order to explore the effects of IC50 concentration on apoptosis induction. Also, Giemsa and DAPI stainings were utilized to explore the morphological changes of cell and nucleus..
Results: IC50 concentration of Salvia extract for A549 cells was determined 5 mg/mL. According to the results, Salvia extract reduced the cell viability of A549 cells. Based on the results, Salvia extract had a significantly greater cytotoxic effect compared to the control sample of A549 cell line. Treatment cells indicated some clear and dose-dependent differences at different times. The results of stainings proved the apoptosis induction in the treatment group. Furthermore, regarding the enzyme expression results the activity of superoxide dismutase and catalase enzymes in the cell groups treated by Salvia extract was significantly increased, compared to the control group.
Conclusion: Consistent with the results of this study, in addition to the anti-oxidative activity Salvia officinalis hydroalcoholic extract revealed significant apoptotic effects on lung cancer cells, considering a time and dose-dependent method.
 

Abstract Aim: The purpose of this research was aimed to evaluate the protective effects of crocin, as an antioxidant agent on Mast cells, blood vessels and biochemical changes of ovary and serum in Busulfan-induced oxidative stress in Mice.                                                                                                                                
Material and Methods: Thirty mature 6-8 weeks aged female NMRI mice in the weight of 22-25 g were randomly divided into 6 groups, and treated for 21 days. The control group only received solvent of Busulfan (BSF) (0.1 ml) intraperitoneally, and BSF group received only Busulfan (10 mgkg^-1, IP/single dose). The experimental groups no. 1, 2, 3 received BSF (10 mgkg^-1 /single dose) with crocin (100, 200, 400 mgkg^-1 /day, IP) and positive group only received crocin (400 mgkg^-1, IP/day). At the end of treatment period, animals were euthanized and left ovary were studied for Mast cells, ovary blood vessels, and Serum, and right ovary for biochemical evaluations. Data was subjected to one‒way analysis of variance (ANOVA) and Tukey to determine if significant difference (P≤0.01) existed among the observed results using SPSS.
Results: Busulfan significantly (P≤0.01) increased mast cells and MDA, while decreased ovary blood vessels and SOD rate, significantly (P=0.000) in comparison to control group. However, crocin in all the used doses, especially in the dose of 200 mgkg^-1, significantly decreased the adverse effects of Busulfan.
Conclusion: The results indicated that crocin can protect ovaries against Busulfan induced damages, and it can be considered as a suitable drug for reducing the toxic effects of Busulfan in chemotherapy.
 

Abstract -
Aim: The purpose of this study was to investigate the protective effects of quercetin on myocardial tissue damage in male Wistar rats during malathion poisoning.
Material and Methods: This study was performed on seven groups of six male rats. After 24 hours of intraperitoneal injection of quercetin, Malathion or a combination of these drugs, the heart of the animals was separated, and tissue slice was prepared. After tissue homogenization, oxidative stress parameters were measured in this area.
Results: Intraperitoneal injection of 200­ mg/kg of malathion significantly induced lipid peroxidation (p < 0.01) and oxidative stress (p < 0.001) as well as necrosis and inflammation in myocardial tissue. However, intra-peritoneal injection of quercetin (50­mg/kg) reduced malathion-induced toxicity on lipid peroxidation (p < 0.001), oxidative stress (p < 0.05), as well as inflammation and myocardial tissue damage.
Conclusion: The results suggest that quercetin is able to improve myocardial tissue damage and restore the rate of oxidative stress in malathion-treated groups to normal levels.
  

Abstract -
Aim: In this study Nurr1 and GDNF, due to their anti-inflammatory and regenerative effects and Nurr1-mediated regulation of GDNF receptor (Ret) expression, were selected to protect dopaminergic SH-SY5Y cell line against neuroinflammation and toxicity caused by 6-OHDA.
Material and Methods: Recombinant lentiviral vectors carrying Nurr1 and GDNF genes were prepared and transdused to SH-SY5Y and astrocytoma (1321N1) cell lines respectively. Also HEK-293T cells were transfected with plasmid carrying GDNF to overexpress this factor; condition media of transduced astrocytoma and transfected HEK-293T cells were collected and stored. Next, overexpression of mentioned factors was demonstrated by RT-PCR. On the other hand, microglial cells were isolated from neonatal rat brains and induced with LPS to produce neuroinflammatory factors; Inducible expression of them was demonstrated by Griess test and RT-PCR. Condition media of microglia was collected and saved. Finally, SH-SY5Y cells overexpressing Nurr1 were treated with condition media of transduced astrocytoma / transfected HEK-293T and then with condition media of LPS-induced microglia or 6-OHDA toxin.
Results: data from MTT assay showed, SH-SY5Y cells overexpressing Nurr1 or pretreated with GDNF are more resistant to toxicity caused by neuroinflammation and 6-OHDA. Also Nurr1 and GDNF have cooperative effects and give more protection to dopaminergic cells.
Conclusion: Nurr1 and GDNF each have protective effects on dopaminergic neural cells against inflammatory factors or 6-OHDA. Also they synergize with each other leading to more protection for dopaminergic neural cells.

Abstract Aim: The aim of this study is to compare sperm parameters (concentration, motility and morphology), DNA damage, percentage and intensity of ROS (Reactive Oxygen Species) and relationship between these parameters in the fertile and infertile men with varicocele.
Material and Methods: In this study, the semen samples from 83 individuals with varicocele and 32 fertile men were studied. Sperm parameters according WHO (World Health Organization) guidelines, protamine deficiency (chromomycin A3 staining), sperm DNA damage (TUNEL assay), percentage and intensity of ROS (DCFH-DA staining) were assessed in fertile and infertile men with varicocele.
Results: The results of this study showed that sperm concentration, percentage of motility and normal morphology were significantly lower in individuals with varicocele compared to fertile individuals. In addition, percentage of sperm DNA damage, protamine deficiency and ROS positive were significantly higher in infertile men with varicocele.
Conclusion: The increase of stress oxidative and testicular temperature in infertile men with varicocele, lead to decrease the quality of sperm parameters as well as negative effects on sperm chromatin integrity. Therefore the process of spermatogenesis and the rate of fertility were affected in these individuals.