Deng Li, Zhou Ling, Zhu Yan, Fan Guangbi, Tang Huajun, Zheng Yujie, Gao Xiaoqing, Guo Kan, Zhou Peng, Yang Chaoxian
Department of Anatomy, College of Basic Medicine, Southwest Medical University, Luzhou, 646000, People's Republic of China.
Clinical Skills Center, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, People's Republic of China.
Stem Cell Rev Rep. 2022 Feb;18(2):570-584. doi: 10.1007/s12015-021-10144-8. Epub 2021 Mar 4.
Previous studies have showed the beneficial effects of mesenchymal stem cells (MSCs) on experimental intracerebral hemorrhage (ICH) animal. Enhancement of the treatment efficacy of MSCs in ICH is essential, considering the diseases association with high rates of disability and mortality. Some auxiliary methods to enhance the beneficial efficacy of MSCs have been introduced. However, the effect of electroacupuncture (EA) on the therapeutic efficacy of MSCs transplantation in hemorrhagic stroke and its potential mechanism is not explored.
ICH rat models were established using collagenase and heparin. 48 h after ICH induction, the rats were randomly divided into model control (MC), MSCs transplantation (MSCs), EA stimulation (EA) and MSCs transplantation combined with EA stimulation (MSCs + EA) groups. We used mNSS test and gait analysis to assess neurological function of rats, and PET/CT to evaluate the volume of hemorrhage focus and level of glucose uptake. The concentrations of MDA, SOD, NSE, S100B and MBP in serum or plasma were examined with ELISA. Neural differentiation of MSCs, and the expressions of Bcl-2, Bax, Arg-1 and iNOS proteins around hematoma were detected by immunofluorescence and immunohistochemistry staining respectively. Western blot was carried out to analyze the expression levels of COX4, OGDH, PDH-E1α, Bcl-2 and Bax proteins. TUNEL staining was used to estimate cell apoptosis and transmission electron microscopy (TEM) was used to observe the ultrastructure and number of mitochondria.
Our data showed that EA promoted neuron-like differentiation of transplanted MSCs and the expressions of BDNF and NGF proteins in ICH rats. The score of mNSS and the gait analysis showed that the recovery of the neurological function in the MSCs + EA group was better than that in the MSCs and EA groups. EA improved the structure of brain tissue, and alleviated brain injury further after MSCs transplantation in ICH rats. When compared with the MSCs and EA groups, the level of glucose uptake and numbers of mitochondria and Arg-1 positive cells in MSCs + EA group increased significantly, but the numbers of apoptotic cells and iNOS positive cells and volume of hemorrhage focus reduced. The expressional levels of COX4, OGDH, PDH-E1α and Bcl-2 proteins increased, while the expressional level of Bax protein decreased compared with those in the MSCs and EA groups.
Our results reveal that EA improve therapeutic efficacy of MSCs transplantation in ICH rats.
先前的研究已表明间充质干细胞（MSCs）对实验性脑出血（ICH）动物具有有益作用。鉴于ICH与高致残率和死亡率相关，提高MSCs在ICH中的治疗效果至关重要。已经引入了一些辅助方法来增强MSCs的有益效果。然而，电针（EA）对出血性中风中MSCs移植治疗效果及其潜在机制的影响尚未得到探索。
使用胶原酶和肝素建立ICH大鼠模型。ICH诱导后48小时，将大鼠随机分为模型对照组（MC）、MSCs移植组（MSCs）、EA刺激组（EA）和MSCs移植联合EA刺激组（MSCs＋EA）。我们使用改良神经功能缺损评分（mNSS）测试和步态分析来评估大鼠的神经功能，并使用PET/CT评估出血灶体积和葡萄糖摄取水平。用酶联免疫吸附测定（ELISA）检测血清或血浆中丙二醛（MDA）、超氧化物歧化酶（SOD）、神经元特异性烯醇化酶（NSE）、S100B蛋白和髓鞘碱性蛋白（MBP）的浓度。分别通过免疫荧光和免疫组织化学染色检测MSCs的神经分化以及血肿周围Bcl-2、Bax、精氨酸酶-1（Arg-1）和诱导型一氧化氮合酶（iNOS）蛋白的表达。进行蛋白质免疫印迹法分析细胞色素C氧化酶4（COX4）、氧化戊二酸脱氢酶（OGDH）、丙酮酸脱氢酶E1α（PDH-E1α）、Bcl-2和Bax蛋白的表达水平。用TUNEL染色估计细胞凋亡，并使用透射电子显微镜（TEM）观察线粒体的超微结构和数量。
我们的数据表明，EA促进了ICH大鼠移植MSCs的神经元样分化以及脑源性神经营养因子（BDNF）和神经生长因子（NGF）蛋白的表达。mNSS评分和步态分析表明，MSCs＋EA组的神经功能恢复优于MSCs组和EA组。EA改善了脑组织结构，并在ICH大鼠MSCs移植后进一步减轻了脑损伤。与MSCs组和EA组相比，MSCs＋EA组的葡萄糖摄取水平、线粒体数量和Arg-1阳性细胞数量显著增加，但凋亡细胞数量、iNOS阳性细胞数量和出血灶体积减少。与MSCs组和EA组相比，COX4、OGDH、PDH-E1α和Bcl-2蛋白的表达水平升高，而Bax蛋白的表达水平降低。
我们的结果表明，EA提高了ICH大鼠MSCs移植的治疗效果。
