Effects of hUCB‑MSCs on recovery of neurological function and TERT expression in brain tissue of rats with cerebral ischemia‑reperfusion injury
- Xiaohui Huang
- Shuangli Zhang
- Fuchun Li
- Yuyun Zhou
- Xiaohe Wang
- Guojiao Fu
- Xueling Ma
- Published online on: October 10, 2017 https://doi.org/10.3892/etm.2017.5274
- Pages: 5843-5846
Copyright: © Huang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
With the incidence of ischemic encephalopathy gradually increased, people have payed increased attention to the prevention and treatment of cerebral ischemia-reperfusion injury (1,2). Cerebral infarction is a common cause of ischemic encephalopathy, thrombolytic therapy after cerebral infarction in time can save the lives of patients, but also may cause ischemia-reperfusion injury. Therefore, if the mechanism of ischemia-reperfusion injury is clear, it is of great significance for timely target treatment for ischemia-reperfusion injury. In recent years, there were more studies on ischemia-reperfusion injury, but there is still not a unified opinion on the prevention and treatment of ischemia-reperfusion injury. Studies showed that the expression of telomerase was increased in the brain tissues of patients with ischemia-reperfusion injury (2,3), while some researchers found that telomerase was overexpressed after the injury of brain tissue, considering the protective effect of telomerase on ischemia-reperfusion injury in order to reduce the tissue damage (4). Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) can secrete a variety of cytokines and neurotrophic factors and promote nerve regeneration, but the conventional route of administration can not fully play the role of hUCB-MSCs. So some scholars proposed nasal administration of hUCB-MSCs to improve neurological function in rats with ischemia-reperfusion injury (5). At present, the study of hUCB-MSCs on nerve injury in rats with ischemia-reperfusion injury is scarce. So the aim of our study was to investigate the effects of hUCB-MSCs on rats with ischemia-reperfusion injury, in order to explore the mechanism of the prevention and treatment of ischemia-reperfusion injury.
Materials and methods
Experiments were performed using 100 healthy adult Wistar rats weighing 180–220 g, provided by College Animal Center, Harbin Medical University. Rats were kept in the animal house under constant temperature (18–25°C) and humidity (60 to 70%) on natural light with C060 sterilized commercial feed and free water intake, with 12 h before fasting. The study was approved by the Ethics Committee of Harbin Medical University.
All rats were randomly divided into control group and observation group and treated with 10% chloral hydrate (3.5 ml/kg) at RT. After anesthesia, the middle cerebral artery ischemia-reperfusion model was established by the suture method, and the specific modeling method was according to the method given by Chopp and Li (6). Then the rats in the observation group were treated with hUCB-MSCs (10 ml/kg) by nasal administration for 14 days, with the saline in the control group. The neurological deficit score and foot fault test were evaluated at 1, 7 and 14 days after treatment, and the rats were sacrificed at 14 days to detect the expression of telomerase reverse transcriptase (TERT) in brain tissue.
The source of hUCB-MSCs: 30 ml human umbilical cord blood was taken under sterile conditions with heparin anticoagulant, then transferred into a 15 ml centrifuge tube and centrifuged at 2,800 × g for 20 min. The upper plasma was removed, collecting the white blood cells in interface layer, and stored at 4°C. The expression of TERT was detected by western blot analysis. The above operations are carried out by the researchers in our laboratory according to the instructions, and the reagents and kits were purchased from LanPai Biotechnology Co., Ltd. (Shanghai, China).
Observation and evaluation of indicators
The criteria for mNSS (7)
The four aspects as sensory, motor, reflex and muscle tensionin of rats were scored with the total score of 18. The higher the score, the more serious the nerve damage.
Foot fault test
The foot fault test was used to evaluate the disorder of rat forelimb, placing the rats on the Hanging mesh grid (2.5×2.5 cm), then recording the total number of bilateral forelimb movement and the number of left forelimb foot errors. The percent of the number of left forelimb foot errors to the total number of bilateral forelimb movement as the statistical results.
The statistical analysis was performed with SPSS 21.0 (IBM Corp., Armonk, NY, USA) and the obtained data are in normal distribution. Measurement data were expressed as mean ± SD and the t-test was performed. Counting data were calculated by Chi-square test. A P-value <0.05 was considered as a significant difference.
Comparison of neurological deficit score between the two groups
There was no significant difference in the scores of mNSS between the two groups before the model establishment (P>0.05), but after the model established there was significant difference in two groups respectively (P<0.05). At 1 day after the operation, the mNSS score of two groups was peaked, which was decreased in the groups with the progress of treatment. The degree of decline in the observation group was significantly greater than that in the control group (P<0.05; Table I).
Comparison of foot fault number between the groups
There was no significant difference in the foot fault number between the two groups before ischemia-reperfusion model establishment (P>0.05), but after the model established the foot fault frequency was significantly increased in each group. At 1 day after operation, the number of foot errors peaked, but it was decreased in the groups with the progress of treatment. The number of foot errors was decreased significantly in the observation group compared to that in the control group (P<0.05; Table II).
The gray value of TERT content in brain tissue in the two groups
The gray value of TERT content in the observation group was 0.983±0.056 LOD, which was significantly higher than that in the control group with the gray value of 0.458±0.052 LOD (P<0.05; Table III).
Comparison of groups of rats with brain tissue immunohistochemical reaction
The H&E staining showed the nucleus or cytoplasm in the brain tissue of rats was brown or yellow brown in the observation group, which showed positive reaction (Fig. 1A). The cytoplasm and the nucleus were stained yellow in the control group, suggested the neurons were shrunken (Fig. 1B).
H&E staining of the rat brain tissue immunohistochemical reaction. (A) The nucleus or cytoplasm was brown or yellow brown in the observation group, which showed positive reaction. (B) The cytoplasm and the nucleus were stained yellow in the control group, suggested the neurons were shrunken.
TERT protein expression in two groups
As Fig. 2 shows, the expression of TERT protein in brain tissue of rats in observation group was significantly decreased compared to that in the control group.
The expression of TERT protein in brain tissue of rats. A, the control group; B, the observation group; TERT, telomerase reverse transcriptase.
Ischemia and hypoxia is an important factor for tissue necrosis. For the brain tissue, short-term ischemia and hypoxia can cause irreversible tissue damage (8). In recent years, with the deep study of ischemic diseases, increased number of studies found that ischemia is not the only factor for the tissue damage (9). Free radical release caused by ischemia-reperfusion plays an important role in the tissue injury induced by ischemia and hypoxia, which is known as ischemia-reperfusion injury. Telomerase refers to an enzyme that is responsible for telomere elongation in cells and is a basic nucleoprotein reverse transcriptase that binds telomere DNA to the eukaryotic chromosome ends. The primary role of telomerase is to maintain telomere integrity and prevents its loss in cell division (10). The activity of telomerase is inhibited in normal cells, but it can be significantly activated in tumor cells. TERT, as a catalytic subunit of telomerase, not only activates telomerase activity, but also protects mitochondria and has an anti-apoptotic effect. Studies showed that TERT can effectively improve neuronal apoptosis and neurological function in patients with ischemia-reperfusion injury (11). Stem cell therapy is a more popular treatment in recent years, which have a good therapeutic effect for some diseases. Some scholars have found that hUCB-MSCs contains a variety of cytokines and nerve growth factors, which can effectively improve neuronal damage, but these growth factors can not pass through the blood-brain barrier (12). Nasal administration not only allows drugs to enter the brain tissue, but also reduces the loss of stem cells.
In this study, there were no significant differences in the scores of mNSS between the two groups before the model establishment (P>0.05), but there was significant differences in two groups after the operation (P<0.05). At 1 day after the operation, the mNSS score of two groups peaked, which was decreased in the groups with the progress of treatment. The degree of decline in the observation group was significantly greater than that in the control group (P<0.05). Similarly, there were no significant differences in the number of preoperative footsteps between the two groups before the model establishment (P>0.05), but there was significant differences in two groups after the operation (P<0.05). At 1 day after the operation, the number of preoperative footsteps was peaked, which was reduced in the groups with the progress of treatment. The degree of reduction in the observation group was significantly greater than that in the control group (P<0.05). The results in our study showed that hUCB-MSCs could effectively reduce the neurological injury of rats with ischemia-reperfusion injury, which was consistent with the decrease of neuronal apoptosis in brain tissue. Zhao et al also found that after administration of hUCB-MSCs, neuronal dysfunction in rats with ischemia-reperfusion injury was improved, which was possibly related to the effects of TERT on promoting astrocyte proliferation and activation, increasing hypoxia ischemic rat brain astrocytes proliferation and promoting neuronal survival (13). With the gray value of the TERT protein as 0.458±0.052 LOD in the control group, which was significantly lower than that in the observation group with 0.983±0.056 LOD, which suggested that hUCB-MSCs could effectively promote the expression of TERT in brain tissue of rats with ischemia-reperfusion. Chen et al also found that hUCB-MSCs significantly increased the expression of TERT protein in rat brain by nasal administration, possibly related to the effect of hUCB-MSCs on promoting the expression of TERT in brain tissue induced by cytokines and neurotrophic factors (14).
In conclusion, hUCB-MSCs can effectively improve the neurological function of cerebral ischemia-reperfusion injury in rats and improve the expression of TERT in brain tissue, which may help to reduce cerebral ischemia-reperfusion injury.
The study was supported by the Post-Doctoral Initial Fund of Heilongjiang Province (LBH-Q15091) and the Distinguished Young Scholars Fund of the Fourth Clinical Medical College of Haerbin Medical University (HYDSYJQ201506).
Lahiani A, Zahavi E, Netzer N, Ofir R, Pinzur L, Raveh S, Arien-Zakay H, Yavin E and Lazarovici P: Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF. Biochim Biophys Acta. 1853:422–430. 2015. View Article : Google Scholar : PubMed/NCBI
Yu H, Chen P, Yang Z, Luo W, Pi M, Wu Y and Wang L: Electro-acupuncture at conception and governor vessels and transplantation of umbilical cord blood-derived mesenchymal stem cells for treating cerebral ischemia/reperfusion injury. Neural Regen Res. 9:84–91. 2014. View Article : Google Scholar : PubMed/NCBI
Guerrero-Beltrán CE, Tapia E, Sánchez-González DJ, Martínez-Martínez CM, Cristobal-García M and Pedraza-Chaverri J: Tert-Butylhydroquinone pretreatment protects kidney from ischemia-reperfusion injury. J Nephrol. 25:84–89. 2012. View Article : Google Scholar : PubMed/NCBI
Khan I, Ali A, Akhter MA, Naeem N, Chotani MA, Iqbal H, Kabir N, Atiq M and Salim A: Epac-Rap1-activated mesenchymal stem cells improve cardiac function in rat model of myocardial infarction. Cardiovasc Ther. 35:352017. View Article : Google Scholar
Chen J, Sanberg PR, Li Y, Wang L, Lu M, Willing AE, Sanchez-Ramos J and Chopp M: Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke. 32:2682–2688. 2001. View Article : Google Scholar : PubMed/NCBI
Luo CJ, Zhang FJ, Zhang L, Geng YQ, Li QG, Hong Q, Fu B, Zhu F, Cui SY, Feng Z, et al: Mesenchymal stem cells ameliorate sepsis-associated acute kidney injury in mice. Shock. 41:123–129. 2014. View Article : Google Scholar : PubMed/NCBI
Sun T, Gao GZ, Li RF, Li X, Li DW, Wu SS, Yeo AE and Jin B: Bone marrow-derived mesenchymal stem cell transplantation ameliorates oxidative stress and restores intestinal mucosal permeability in chemically induced colitis in mice. Am J Transl Res. 7:891–901. 2015.PubMed/NCBI
Chung DJ, Choi CB, Lee SH, Kang EH, Lee JH, Hwang SH, Han H, Lee JH, Choe BY, Lee SY and Kim HY: Intraarterially delivered human umbilical cord blood-derived mesenchymal stem cells in canine cerebral ischemia. J Neurosci Res. 87:3554–3567. 2009. View Article : Google Scholar : PubMed/NCBI
Li J, Qu Y, Chen D, Zhang L, Zhao F, Luo L, Pan L, Hua J and Mu D: The neuroprotective role and mechanisms of TERT in neurons with oxygen-glucose deprivation. Neuroscience. 252:346–358. 2013. View Article : Google Scholar : PubMed/NCBI
Leong KH, Zhou LL, Lin QM, Wang P, Yao L and Huang ZT: Therapeutic effects of various methods of MSC transplantation on cerebral resuscitation following cardiac arrest in rats. Mol Med Rep. 13:3043–3051. 2016. View Article : Google Scholar : PubMed/NCBI
Zhao F, Qu Y, Xiong T, Duan Z, Ye Q and Mu D: The neuroprotective role of TERT via an antiapoptotic mechanism in neonatal rats after hypoxia-ischemia brain injury. Neurosci Lett. 515:39–43. 2012. View Article : Google Scholar : PubMed/NCBI
Chen M, Xiang Z and Cai J: The anti-apoptotic and neuro-protective effects of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) on acute optic nerve injury is transient. Brain Res. 1532:63–75. 2013. View Article : Google Scholar : PubMed/NCBI