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Mohammad Ali Takhshid * 
1, Amir Mahmoodazdeh2 , Sayed Mohammad Shafiee3 , Mohsen Sisakht2 , Zahra Khosdel2
1, Amir Mahmoodazdeh2 , Sayed Mohammad Shafiee3 , Mohsen Sisakht2 , Zahra Khosdel2
1- Professor Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
2- Assistant Professor Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
3- Associated Professor Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
2- Assistant Professor Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
3- Associated Professor Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract:
Purpose: In the present study, the culture of embryonic spinal motor neurons (SMNs) was used to assess the impacts of adrenomedullin (AM) on neurotoxic effects of doxorubicin (DOX).
Materials and methods: To prepare the culture of rat embryonic SMNs, spinal cords were isolated from the rat embryos, digested enzymatically, and triturated to obtain spinal cell suspension. Then, the SMNs were purified from the cell suspension using a single gradient of OptiPrep and were cultured. The SMNs were treated with DOX (0.0-100 µM) and AM (3.125-100 nM) and their viability and apoptosis were evaluated using MTT and annexin V flowcytometric assays. Oxidative stress was assessed through the measurement of cellular reactive oxygen species (ROS), nitric oxide (NO), malondialdehyde (MDA), and 8-iso-prostaglandin F2α (iPF2α) levels. Finally, qPCR was employed to determine the expressions of interleukin1-β (IL-1β), inducible NO synthase (iNOS), tumor necrosis factor-α (TNF-α), SRY-related protein 9 (SOX9), matrix metalloproteinase (MMP)-3 and -13.
Results: The viability of SMNs was decreased following DOX treatment dose-dependently (IC50 = 10.54 µM). DOX increased the cellular ROS, MDA, NO, and iPF2α levels (p<0.001). Additionally, AM reduced DOX-induced cell death dose-dependently (p<0.001). AM (50 nM) pretreatment also reduced the DOX-induced oxidative stress (p<0.01) and -genes expression (p<0.01).
Conclusion: Based on the results, AM might be considered a protective factor against chemotherapy-induced toxicity in SMNs.
Materials and methods: To prepare the culture of rat embryonic SMNs, spinal cords were isolated from the rat embryos, digested enzymatically, and triturated to obtain spinal cell suspension. Then, the SMNs were purified from the cell suspension using a single gradient of OptiPrep and were cultured. The SMNs were treated with DOX (0.0-100 µM) and AM (3.125-100 nM) and their viability and apoptosis were evaluated using MTT and annexin V flowcytometric assays. Oxidative stress was assessed through the measurement of cellular reactive oxygen species (ROS), nitric oxide (NO), malondialdehyde (MDA), and 8-iso-prostaglandin F2α (iPF2α) levels. Finally, qPCR was employed to determine the expressions of interleukin1-β (IL-1β), inducible NO synthase (iNOS), tumor necrosis factor-α (TNF-α), SRY-related protein 9 (SOX9), matrix metalloproteinase (MMP)-3 and -13.
Results: The viability of SMNs was decreased following DOX treatment dose-dependently (IC50 = 10.54 µM). DOX increased the cellular ROS, MDA, NO, and iPF2α levels (p<0.001). Additionally, AM reduced DOX-induced cell death dose-dependently (p<0.001). AM (50 nM) pretreatment also reduced the DOX-induced oxidative stress (p<0.01) and -genes expression (p<0.01).
Conclusion: Based on the results, AM might be considered a protective factor against chemotherapy-induced toxicity in SMNs.
Type of Study: Original |
Subject:
Cellular and molecular Neuroscience
Received: 2021/08/18 | Accepted: 2022/04/12
Received: 2021/08/18 | Accepted: 2022/04/12
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