人脐血间充质干细胞激活磷脂酰肌醇3激酶/蛋白激酶B通路对脑缺血再灌注大鼠的神经保护作用

doi:10.3969/j.issn.1673-5765.2023.11.011

摘要/Abstract

摘要： 目的探究人脐血间充质干细胞（human umbilical cord blood mesenchymal stem cells，hUCBMSCs）调控磷脂酰肌醇3激酶/蛋白激酶B（phosphoinositide3-kinase/protein kinase B，PI3K/Akt）通路对脑缺血再灌注损伤大鼠周细胞表达和神经功能变化的影响。
方法取雄性SD大鼠共72只，随机分组为假手术组、模型组、干细胞组、干细胞+抑制剂组，每组18只。除假手术组外，剩余各组均建立脑缺血再灌注模型。造模成功后，模型组尾静脉注射0.1 mL的磷酸盐缓冲盐溶液（phosphate-buffered saline，PBS），干细胞组尾静脉注射0.1 mL含有2×106个hUCBMSCs的PBS，干细胞＋抑制剂组尾静脉注射0.1 mL含2×106个hUCBMSCs的PBS及腹腔注射PI3K/Akt抑制剂LY294002（0.03 mg/100 g），LY294002每天给药1次，直至处死。术后1、3、7、14 d测定改良神经功能缺损评分（modified neurological severity scores，mNSS）。术后7、14 d，采用2，3，5-氯化三苯基四氮唑（2，3，5-triphenyltetrazolium chloride，TTC）染色法检测大鼠脑梗死面积，尼氏染色检测大脑皮质缺血半暗带正常神经元细胞数，免疫组织化学法检测大脑皮质缺血半暗带血小板衍生生长因子受体-β（platelet derived growth factor receptor-β，PDGFRβ）阳性周细胞的数量，免疫印迹法检测大脑皮质缺血半暗带PI3K/Akt通路相关蛋白p-Akt及Akt的表达。
结果术后7 d，4组各项指标整体差异均有统计学意义（P<0.001）。与假手术组比较，模型组的大鼠正常神经元细胞数[（55.42±4.75）个 vs.（8.50±1.64）个，P<0.001]和p-Akt/Akt（1.00±0.00 vs. 0.47±0.06，P=0.002）均降低，脑PDGFRβ+周细胞数量[（1.08±0.29）个 vs.（13.67±2.47）个，P<0.001]增加；与模型组比较，干细胞组大鼠的mNSS评分[（6.33±0.71）分 vs.（4.78±0.98）分，P<0.001]和脑梗死率（32.66%±1.76% vs. 14.60%±0.52%，P<0.001）均降低，正常神经元细胞数[（8.50±1.64）个 vs.（23.17±1.77）个，P<0.001]、脑PDGFRβ+周细胞数量[（13.67±2.47）个 vs.（28.50±3.19）个，P<0.001]和p-Akt/Akt（0.47±0.06 vs. 0.83±0.18，P=0.017）均增加；与干细胞组比较，干细胞+抑制剂组的大鼠mNSS评分[（4.78±0.98）分 vs.（6.11±0.78）分，P=0.002]和脑梗死率（14.60%±0.52% vs. 27.85%±0.59%，P<0.001）均增加，正常神经元细胞数[（23.17±1.77）个 vs.（11.83±0.88）个，P=0.003]、脑PDGFRβ+周细胞数量[（28.50±3.19）个 vs.（20.33±1.44）个，P=0.007]和p-Akt/Akt（0.83±0.18 vs. 0.36±0.11，P=0.003）均降低。术后14 d，4组各项指标整体差异均有统计学意义（P<0.001）。与假手术组比较，模型组的大鼠正常神经元细胞数[（57.08±3.79）个 vs.（11.25±5.52）个，P<0.001]和p-Akt/Akt（1.00±0.00 vs. 0.53±0.12，P=0.002）均降低，脑PDGFRβ+周细胞数量[（2.00±0.25）个 vs.（13.42±1.04）个，P<0.001]增加；与模型组比较，干细胞组大鼠的mNSS评分[（4.89±0.78）分 vs.（2.33±0.87）分，P<0.001]和脑梗死率（32.58%±1.96% vs. 11.78%±1.92%，P<0.001）均降低，正常神经元细胞数[（11.25±5.52）个 vs.（31.00±1.89）个，P<0.001]、脑PDGFRβ+周细胞数量[（13.42±1.04）个 vs.（31.42±3.66）个，P<0.001]和p-Akt/Akt（0.53±0.12 vs. 0.93±0.12，P=0.004）均增加；与干细胞组比较，干细胞+抑制剂组大鼠的mNSS评分[（2.33±0.87）分 vs.（4.44±0.53）分，P<0.001]和脑梗死率（11.78%±1.92% vs. 25.25%±2.76%，P<0.001）均增加，正常神经元细胞数[（31.00±1.89）个 vs.（13.83±1.04）个，P=0.002]、脑PDGFRβ+周细胞数量[（31.42±3.66）个 vs.（20.67±1.42）个，P<0.001]和p-Akt/Akt（0.93±0.12 vs. 0.53±0.09，P=0.005）均降低。
结论 hUCBMSCs可能通过激活PI3K/Akt通路促进周细胞的存活募集，发挥神经保护的作用，进而促进脑缺血再灌注损伤大鼠的神经功能恢复。

文章导读： 干细胞通过激活PI3K/Akt信号通路，促进周细胞的存活募集，在脑缺血再灌注损伤大鼠中发挥神经保护作用。

关键词: 脐血间充质干细胞; 脑缺血再灌注; PI3K/Akt信号通路; 周细胞

Abstract: Objective To discuss the effect of pericyte expression and the change of neurological function of human umbilical cord blood mesenchymal stem cells (hUCBMSCs) on cerebral ischemia-reperfusion injury rats by regulating phosphoinositide3-kinase/protein kinase B (PI3K/Akt) pathway.
Methods A total of 72 male Sprague Dawley rats were randomly divided into sham group, model group, hUCBMSCs group, hUCBMSCs+inhibitor group, with 18 in each group. Except for sham group, the rat models with cerebral ischemia-reperfusion were established in the other groups. After successful modeling, 0.1 mL phosphate-buffered saline (PBS) was injected into the tail vein of the model group, 0.1 mL PBS containing 2×106 hUCBMSCs was injected into the tail vein of the hUCBMSCs group, 0.1 mL PBS containing 2×106 hUCBMSCs was injected into the tail vein of the hUCBMSCs+inhibitor group and PI3K/Akt inhibitor LY294002 (0.03 mg/100 g) was injected intraperitoneally, LY294002 was administered once a day until death. Modified neurological severity scores (mNSS) were measured at 1, 3, 7 and 14 days after operation. After 7 and 14 days of operation, the area of cerebral infarction was measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The number of normal neurons in ischemic penumbra of cerebral cortex were detected by Nissl staining. The number of platelet derived growth factor receptor-β+ (PDGFRβ+) pericytes in ischemic penumbra of cerebral cortex were detected by immunohistochemistry. The expressions of PI3K/Akt pathway related proteins p-Akt and Akt in ischemic penumbra of cerebral cortex were detected by Western Blot.
Results On the 7th day after operation, there were significant differences in all indexes among the four groups (P<0.001). As compared with sham group, the number of normal neurons (55.42±4.75 vs. 8.50±1.64, P<0.001) and p-Akt/Akt (1.00±0.00 vs. 0.47±0.06, P=0.002) in model group were decreased, however the number of PDGFRβ+ pericytes (1.08±0.29 vs. 13.67±2.47, P<0.001) were increased. As compared with model group, the mNSS (6.33±0.71 vs. 4.78±0.98, P<0.001) and the rate of cerebral infarction (32.66%±1.76% vs. 14.60%±0.52%, P<0.001) in hUCBMSCs group were decreased; however the number of normal neurons (8.50±1.64 vs. 23.17±1.77, P<0.001), the number of PDGFRβ+ pericytes (13.67±2.47 vs. 28.50±3.19, P<0.001) and p-Akt/Akt (0.47±0.06 vs. 0.83±0.18, P=0.017) were increased. As compared with hUCBMSCs group, the mNSS (4.78±0.98 vs. 6.11±0.78, P=0.002) and the rate of cerebral infarction (14.60%±0.52% vs. 27.85%±0.59%, P<0.001) in hUCBMSCs+inhibitor group were increased; however the number of normal neurons (23.17±1.77 vs. 11.83±0.88, P=0.003), the number of PDGFRβ+ pericytes (28.50±3.19 vs. 20.33±1.44, P=0.007) and p-Akt/Akt (0.83±0.18 vs. 0.36±0.11, P=0.003) were decreased. On the 14th day after operation, there were significant differences in all indexes among the four groups (P<0.001). As compared with sham group, the number of normal neurons (57.08±3.79 vs. 11.25±5.52, P<0.001) and p-Akt/Akt (1.00±0.00 vs. 0.53±0.12, P=0.002) in model group were decreased, however the number of PDGFRβ+ pericytes (2.00±0.25 vs. 13.42±1.04, P<0.001) were increased. As compared with model group, the mNSS (4.89±0.78 vs. 2.33±0.87, P<0.001) and the rate of cerebral infarction (32.58%±1.96% vs. 11.78%±1.92%, P<0.001) in hUCBMSCs group were decreased; however the number of normal neurons (11.25±5.52 vs. 31.00±1.89, P<0.001), the number of PDGFRβ+ pericytes (13.42±1.04 vs. 31.42±3.66, P<0.001) and p-Akt/Akt (0.53±0.12 vs. 0.93±0.12, P=0.004) were increased. As compared with hUCBMSCs group, the mNSS (2.33±0.87 vs. 4.44±0.53, P<0.001) and the rate of cerebral infarction (11.78%±1.92% vs. 25.25%±2.76%, P<0.001) in hUCBMSCs+inhibitor group were increased; however the number of normal neurons (31.00±1.89 vs. 13.83±1.04, P=0.002), the number of PDGFRβ+ pericytes (31.42±3.66 vs. 20.67±1.42, P<0.001) and p-Akt/Akt (0.93±0.12 vs. 0.53±0.09, P=0.005) were decreased.
Conclusions hUCBMSCs may play a neuroprotective role by promoting the survival and recruitment of pericytes through the activation of PI3K/Akt pathway, and then promote the recovery of neurological function in rats with cerebral ischemia-reperfusion injury.

Key words: Umbilical cord blood mesenchymal stem cells; Cerebral ischemia-reperfusion; PI3K/Akt signaling pathway; Pericytes

范金金, 丁立, 张跃亮, 陈俊, 张贝, 李雪清, 佟旭, 王云甫, 艾志兵. 人脐血间充质干细胞激活磷脂酰肌醇3激酶/蛋白激酶B通路对脑缺血再灌注大鼠的神经保护作用[J]. 中国卒中杂志, 2023, 18(11): 1289-1297.

FAN Jinjin, DING Li, ZHANG Yueliang, CHEN Jun, ZHANG Bei, LI Xueqing, TONG Xu, WANG Yunfu, AI Zhibing. The Neuroprotective Effects of Human Umbilical Cord Blood Mesenchymal Stem Cells on Cerebral Ischemia-Reperfusion Rats by Activating PI3K/Akt Pathway[J]. Chinese Journal of Stroke, 2023, 18(11): 1289-1297.

参考文献

[1] SMITH M，REDDY U，ROBBA C，et al. Acute ischaemic stroke：challenges for the intensivist[J]. Intensive Care Med，2019，45（9）：1177-1189.
[2] GONZALES A L，KLUG N R，MOSHKFOROUSH A，et al. Contractile pericytes determine the direction of blood flow at capillary junctions[J]. Proc Natl Acad Sci U S A，2020，117（43）：27022-27033.
[3] GAUTAM J，YAO Y. Roles of pericytes in stroke pathogenesis[J]. Cell Transplant，2018，27（12）：1798-1808.
[4] DANEMAN R，ZHOU L，KEBEDE A A，et al. Pericytes are required for blood-brain barrier integrity during embryogenesis[J]. Nature，2010，468（7323）：562-566.
[5] LI J S，ZHANG Q，WANG W，et al. Mesenchymal stem cell therapy for ischemic stroke：a look into treatment mechanism and therapeutic potential[J]. J Neurol，2021，268（11）：4095-4107.
[6] HE J L，LIU J，HUANG Y Y，et al. Oxidative stress，inflammation，and autophagy：potential targets of mesenchymal stem cells-based therapies in ischemic stroke[J/OL]. Front Neurosci，2021，15：641157[2022-09-15]. https://doi.org/10.3389/fnins.2021.641157.
[7] XU L M，JI H F，JIANG Y F，et al. Exosomes derived
from CircAkap7-modified adipose-derived mesenchymal
stem cells protect against cerebral ischemic injury[J/OL]. Front Cell Dev Biol，2020，8：569977[2022-09-15]. https://doi.org/10.3389/fcell.2020.569977.
[8] KHAN H，SINGH A，THAPA K，et al. Therapeutic modulation of the phosphatidylinositol 3-kinases
（PI3K）pathway in cerebral ischemic injury[J/OL]. Brain Res，2021：147399[2022-09-15]. https://doi.org/10.1016/j.brainres.2021.147399.
[9] SUN X，HUANG L Y，PAN H X，et al. Bone marrow mesenchymal stem cells and exercise restore motor function following spinal cord injury by activating PI3K/AKT/mTOR pathway[J]. Neural Regen Res，2023，18（5）：1067-1075.
[10] ZHANG Y H，ZHANG Y，BAI Y，et al. Involvement of PUMA in pericyte migration induced by methamphetamine[J]. Exp Cell Res，2017，356（1）：28-39.
[11] UEBELHOER M，NÄTYNKI M，KANGAS J，et al.
Venous malformation-causative TIE2 mutations mediate an AKT-dependent decrease in PDGFB[J]. Hum Mol Genet，2013，22（17）：3438-3448.
[12] ASHWAL S，TONE B，TIAN H R，et al. Core and penumbral nitric oxide synthase activity during cerebral ischemia and reperfusion[J]. Stroke，1998，29（5）：1037-1046，1047.
[13] TANG Y W，YU P，CHENG L. Current progress in the derivation and therapeutic application of neural stem cells[J/OL]. Cell Death Dis，2017，8（10）：e3108[2022-09-15]. https://doi.org/10.1038/cddis.2017.504.
[14] HAO L，ZOU Z M，TIAN H，et al. Stem cell-based therapies for ischemic stroke[J/OL]. Biomed Res Int，2014，2014：468748[2022-09-15]. https://doi.org/10.1155/2014/468748.
[15] YE Y C，CHANG Z H，WANG P，et al. Infarct-preconditioning exosomes of umbilical cord mesenchymal stem cells promoted vascular remodeling and neurological recovery after stroke in rats[J]. Stem Cell Res Ther，2022，13（1）：378.
[16] TACHIBANA M，AGO T，WAKISAKA Y，et al. Early reperfusion after brain ischemia has beneficial effects beyond rescuing neurons[J]. Stroke，2017，48（8）：2222-2230.
[17] NAKAMURA K，ARIMURA K，NISHIMURA A，
et al. Possible involvement of basic FGF in the upregulation of PDGFRβ in pericytes after ischemic stroke[J/OL]. Brain Res，2016，1630：98-108[2022-09-15]. https://doi.org/10.1016/j.brainres.2015.11.003.
[18] SUN J Q，HUANG Y N，GONG J，et al. Transplantation of hPSC-derived pericyte-like cells promotes functional recovery in ischemic stroke mice[J]. Nat Commun，2020，11（1）：5196.
[19] NAKAMURA K，IKEUCHI T，NARA K，et al.
Perlecan regulates pericyte dynamics in the maintenance and repair of the blood-brain barrier[J]. J Cell Biol，2019，218（10）：3506-3525.
[20] WANG H J，RAN H F，YIN Y，et al. Catalpol improves impaired neurovascular unit in ischemic stroke rats via enhancing VEGF-PI3K/AKT and VEGF-MEK1/2/ERK1/2 signaling[J]. Acta Pharmacol Sin，2022，43（7）：1670-1685.
[21] CAI J L，LIANG J Y，ZHANG Y T，et al. Cyclo-
（Phe-Tyr）as a novel cyclic dipeptide compound alleviates ischemic/reperfusion brain injury via JUNB/JNK/NF-κB and SOX5/PI3K/AKT pathways[J/OL]. Pharmacol Res，2022，180：106230[2022-09-15]. https://doi.org/10.1016/j.phrs. 2022.106230.
[22] TRENKWALDER T，DEINDL E，BONGIOVANNI D，et al. Thymosin-β4-mediated therapeutic neovascularization：role of the PI3K/AKT pathway[J/OL]. Expert Opin Biol Ther，2015，15 Suppl 1：S175-S185[2022-09-15]. https://doi.org/10.1517/14712598.2015.1011122.
[23] HARIBALAGANESH R，SHEIKPRANBABU S，ELAYAPPAN B，et al. Pigment-epithelium-derived factor down regulates hyperglycemia-induced apoptosis via PI3K/Akt activation in goat retinal pericytes[J]. Angiogenesis，2009，12（4）：381-389.
[24] HU E，HU W，YANG A L，et al. Thrombin promotes pericyte coverage by Tie2 activation in a rat model of intracerebral hemorrhage[J]. Brain Res，2019，1708：58-68.