Volume 31, Issue 4 (1-2023)                   JGUMS 2023, 31(4): 350-361 | Back to browse issues page

Research code: 162369184
Ethics code: IR.IAU.TABRIZ.REC.1400.115


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Miri S Z, Rahmani Kahnamoei J, Safavi Khalkhali S E. Effect of Human Mesenchymal Stem Cell-Conditioned Medium on Oxidative Stress Induced by Carbon Tetrachloride in the Liver Tissue of Rats. JGUMS 2023; 31 (4) :350-361
URL: http://journal.gums.ac.ir/article-1-2465-en.html
1- Graduate of Veterinary Medicine, Faculty of Veterinary, Tabriz Branch Medical Sciences, Islamic Azad University, Tabriz, Iran.
2- Department of Clinical Science, Faculty of Veterinary, Tabriz Branch Medical Sciences, Islamic Azad University, Tabriz, Iran. , jrahmani28@gmail.com
3- Department of Basic Science, Faculty of Veterinary, Tabriz Branch Medical Sciences, Islamic Azad University, Tabriz, Iran.
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Introduction
As the body’s largest internal organ, the liver plays an essential role in many physiological processes. It is sensitive and vulnerable to various types of toxins and microbes [1]. Many drugs cause the production of free radicals. If the amount of these radicals be more than the antioxidant capacity, they can cause damage to biomolecules such as lipids of liver cell membrane [2]. Liver transplantation is a treatment method for liver failure [3]. However, liver transplantation has limitations such as the shortage of suitable donors [45, 6, 7]. Another method for treatment of liver failure is the use of stem cells [8]. However, the direct use of mesenchymal stem cells has some problems such as the stimulation of the immune system [9]. Therefore, it seems necessary to find alternative methods. In this study, we use product that include factors secreted from stem cells without the presence of stem cells themselves so that we can avoid limitations such as conditioned medium. We aim to investigate the healing effect of a conditioned medium obtained from umbilical cord stem cells on liver damage.
Methods
The study was conducted in two stages. The first stage included the preparation of human mesenchymal stem cell-conditioned medium (hMSC-CM) in the Biotechnology Research Center of Tabriz Branch, Islamic Azad University. In the second stage, we assessed the effect of conditioned medium on the total antioxidant capacity (TAC) and malondialdehyde (MDA). In this study, 21 adult male Wistar rats were used, which were obtained from the Laboratory Animal Breeding Center of Islamic Azad University, Tabriz branch. Rats were randomly divided into three groups of 7 including control group, treatment group A received carbon tetrachloride (CCI4), and treatment group B received CCI4 and hMSC-CM under normal maintenance conditions. The control group received normal saline in a single dose of 2 cc/kg intraperitoneally. Treatment groups A and B received intraperitoneal CCI4 at a dose of 2 ml/kg body weight and olive oil with a ratio of 1:1 [11, 12]. Twenty-four hours after CCI4 injection for three consecutive days, the rats in group B were treated by injection of hMSC-CM [13]. Twenty-four hours after the second injection of hMSC-CM, blood samples were collected from all rats, and oxidative stress indicators (TAM and MDA) were evaluated by diagnostic kits; TAM was evaluated by the Ferric Reducing Ability of Plasma (FRAP) method and MDA by the ThioBarbituric Acid Reactive Substances (TBARS) method. One-way ANOVA followed by Tukey’s post hoc test was used to compare the means between different groups. Data analysis was carried out in SPSS version 25 software (IBM Corp., Armonk, NY, USA).
Results
As shown in Table 1 and Figure 1, there were no significant differences between groups in TAC (P=0.175) and MDA level (P=0.478).


As a result, hMSC-CM had no significant effect on treating liver damage caused by CCI4 injection. The lowest amount of TAC belonged to the group treated with CCI4 (0.46 mmol/L), while the highest TAC was observed in the group treated with CCI4 and hMSC-CM (0.65 mmol/L). The lowest amount of MDA was seen in the control group (2.20 μmol/L), while the highest amount was in the group treated with CCI4 (2.61 μmol/L).
Discussion
Since the antioxidant effects of hMSC-CM have been confirmed in previous studies [14], this paper aimed to investigate its effects on CCI4-induced oxidative stress. In Ogali et al.’s study, the toxicity induced by CCI4 caused a significant reduction in TAC of animals [15]. Since the CCI4-induced oxidative stress is created through the production of reactive oxygen species (ROS), a part of the TAC of the tissue is used to deal with the oxides caused by ROS. Therefore, reduced TAC in CCI4-treated animals is reasonable [1617].
MDA is one of the most common oxidative stress markers [18]. The results of our study, consistent with the studies by Mohammadalipour et al. [19] and Okda et al. [20], showed an increased level of MDA in the group treated with CCI4. Considering the effect of CCI4 on ROS production, a high level of MDA was expected. Considering the role of antioxidant enzymes in dealing with ROS as well as the results obtained in the present study, it can be said that the use of hMSC-CM may treat oxidative stress by increasing the activity of antioxidant enzymes [21]. Amelioration of oxidative stress can reduce CCI4-induced liver lesions. According to the study by Bahmani et al. [22], treatment with hMSC-CM may lead to improvement of antioxidant indices, but according to the present study, the duration of hMSC-CM injection and the interval between injections are influential factors of the significance of the result and recovery. It can be concluded that injection of hMSC-CM within 24 hours after causing liver damage does not have significant effects on TAC and MDA level. Different results may be obtained by increasing the duration of treatment or increasing the number of stem cells from which the medium was prepared.

Ethical Considerations
Compliance with ethical guidelines

This study was conducted according (to) the principles of the ethical care and use of (laboratory) animals, and obtained ethical approval (Code: IAU.TABRIZ.REC.1400.115).

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
Conceptualization and design: Jafar Rahmani Kahnamoei; Acquisition, analysis, or interpretation of data: Jafar Rahmani Kahnamoei and Seyyedeh Zahra; Drafting of the manuscript: Seyyedeh Zahra Miri; Critical revision of the manuscript for important intellectual content: Jafar Rahmani Kahnamoei; Supervision: Jafar Rahmani Kahnamoei and Seyyed Esmaeil Safavi Khalkhali.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors would like to thank the Pathology Laboratory of Tabriz Branch, Islamic Azad University for their coopration.
 
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Review Paper: Applicable | Subject: Special
Received: 2022/01/25 | Accepted: 2022/08/23 | Published: 2022/11/16

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