RESUMO
Previous studies have shown that the combination of mesenchymal stem cell (MSC) transplantation and electroacupuncture (EA) stimulation is a neuroprotective strategy for treating intracerebral hemorrhage (ICH). However, the underlying mechanisms by which the combined treatment promotes neuroprotection remain unclear. This study was designed to investigate the effects of the combined treatment on synaptic plasticity and elucidate their underlying mechanisms. Therefore, rat ICH models were established by injecting collagenase and heparin, and the animals were randomly divided into model control (MC), EA stimulation (EA), MSC-derived neuron-like cell transplantation (MSC-dNLCs), and MSC-dNLC transplantation combined with EA stimulation (MSC-dNLCs+EA) groups. We observed the ultrastructure of the brain and measured the brain water content (BWC) and the levels of the microtubule-associated protein 2 (MAP2), galactocerebrosidase (GALC), and glial fibrillary acidic protein (GFAP) proteins. We also measured the levels of the phosphorylated mammalian target of rapamycin (mTOR) and 70 kDa ribosomal protein S6 kinase (p70S6K) proteins, as well as the expression of synapse-related proteins. The BWC increased in rats after ICH and decreased significantly in ICH rats treated with MSC-dNLC transplantation, EA stimulation, or combined therapy. Meanwhile, after ICH, the number of blood vessels increased more evidently, but only the combined treatment reduced the number of blood vessels among rats receiving the three treatments. Moreover, the levels of MAP2, GALC, postsynaptic density 95 (PSD95), and synaptophysin (SYP) proteins, as well as the levels of the phosphorylated mTOR and p70S6k proteins, increased in the MSC-dNLCs+EA group compared with those in the MSC-dNLCs and EA groups. Compared with the MC group, GFAP expression was significantly reduced in the MSC-dNLCs, EA, and MSC-dNLCs+EA groups, but the differences among the three treatment groups were not significant. In addition, the number of synapses increased only in the MSC-dNLCs+EA group compared to the MC group. Based on these data, the combination of MSC-dNLC transplantation and EA stimulation exerts a synergistic effect on improving the consequences of ICH by relieving cerebral edema and glial scarring, promoting the survival of neurons and oligodendrocytes, and activating mTOR/p70S6K signaling to enhance synaptic plasticity.
RESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our results reveal that EA improve therapeutic efficacy of MSCs transplantation in ICH rats.