دانشگاه اراک سلول و بافت 2228-7035 16 4 2025 12 22 Curcumin Reinstates Osteogenic Differentiation Disrupted by DEHP in Rat Mesenchymal Stem Cells کورکومین تمایز استئوژنیک مختل‌شده توسط DEHP را در سلول‌های بنیادی مزانشیم رت بازسازی می‌کند 369 388 730596 10.66224/JCT.16.4.369 FA محمدحسین آبنوسی دانشگاه اراک، دانشکده علوم پایه، گروه زیست شناسی 0000-0001-6655-1890 محجوبه لک گروه زیست شناسی، دانشکده علوم پایه، دانشگاه اراک، اراک، ایران 0009-0000-9158-8687 Journal Article 2025 07 21 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. هدف: دی-2-اتیلهگزیل فتالات (DEHP) به‌عنوان نرم‌کننده در محصولات PVC پزشکی استفاده می‌شود و به‌دلیل عدم اتصال کووالانسی، در فرایند درمان جدا شده و موجب آلودگی مغز استخوان می‌شود. مطالعات نشان داده‌اند که DEHP با القای استرس اکسیداتیو، تمایز استئوژنیک سلول‌های بنیادی مزانشیمی (BMSCs) را مهار می‌کند. در این پژوهش، نقش آنتی‌اکسیدانتی کورکومین (Cur) در کاهش آسیب‌های DEHP بررسی شده است. مواد و روش‌ها: سلول‌ها BMSC از رت ویستار استخراج و پس از پاساژ سوم، به‌مدت ۲۱ روز در محیط استئوژنیک با DEHP (100 μM)، کورکومین (0.1 μM) و ترکیب هم‫زمان آن‫ها تیمار شدند. توانائی زیستی و تمایز استئوژنیک با آزمون‌های آلیزارین رد، کلسیم ماتریکس و آلکالین فسفاتاز ارزیابی شد. استرس اکسیداتیو با اندازه‌گیری MDA، فعالیت CAT و SOD و سطح TAC بررسی گردید. همچنین بیان ژن‌های استئوژنیک از طریق RT-PCR برای ژن‌های SMAD1، BMP2، BMP7، RUNX2، COL-1، ALP، OC و GAPDH انجام شد. نتایج: DEHP موجب کاهش شدید توانائی زیستی و تمایز استئوژنیک BMSCs شد (p<0.0001)، در حالی‫که کورکومین در تیمار هم‫زمان این اثرات را به‌طور معنی‌دار جبران نمود، به‌طوری‌که ماتریکس استخوانی، غلظت کلسیم و فعالیت آلکالین فسفاتاز به سطح کنترل رسید. همچنین بیان همه ژن‌های دخیل در تمایز استئوژنیک افزایش یافت و Cur موجب کاهش MDA (p<0.05) و افزایش فعالیت CAT، SOD و TAC شد، و توانست اثرات استرس اکسیداتیو ناشی از DEHP را خنثی کند. نتیجه‌گیری: کورکومین با مهار استرس اکسیداتیو توانست اثرات مهاری DEHP بر تمایز استئوژنیک BMSCs را جبران نماید. لذا پیشنهاد می‌شود درصورت مواجهه با DEHP در طی درمان دراز مدت از کورکومین استفاده شود.

https://jct.araku.ac.ir/article_730596_1aa57dc96cf7389f98a92b839e1fbb39.pdf