| Ischemic stroke is one of the most common cerebrovascular diseases with high mortality and disability rates. The active prevention and treatment of ischemic stroke to reduce mortality and severe disability rate has great social significance. Apart from intravenous delivery of tissue plasminogen activator during the first hours after an ischemic stroke, only a few percent of patients could receive this treatment, there is no treatment proven to be effective in improving functional recovery after the ischemic phase. Stem cell transplantation offers an exciting new therapeutic avenue to restore function after ischemic stroke. Because, there are no ethical concerns with BMSCs, they are easy obtained and highly expandable without ethical or immunological problems. BMSCs seem to be a promising source for stem cell transplantation. Transplanted BMSCs can be integrated into the ischemic boundary zone through secreting soluble paracrine factors or directly interacting with parenchymal cells to display beneficial effects, such as cell replacement, neuroprotection, angiogenesis, neuroinflammation and so on. Recent years, more and more researchers reveal the mechanisms of BMSCs transplantation therapy from the immunoregulatory and paracrine effects. To confirm that the treatment of ischemic stroke model with transplanted BMSCs through paracrine effect, at the acute of ischemic stroke, BMSCs and their paracrine factors (BMSCs supernatant) were injected to the lesions. After 7 days, cerebral infarction area and neurological recovery were assessed. The results showed that the injection of paracrine factor has a similar therapeutic effect with transplanted BMSCs. Astrocytes take up the majority of non-neuronal cells in the central nervous system, which play an important role in physiological and pathological conditions of the central nervous system. Dysfunction of astrocytes led by ischemic insult can remarkably influence the generation, progress and prognosis of ischemic stroke, which can cause cell apoptosis, releasing inflammatory cytokines, glial scar formation, water balance disorders, and glutamate metabolism. Thus, in this study, we further investigated the effects of paracrine factors on the activity and reactive astrogliosis of injured astrocytes induced by ischemic stroke in vivo. The results showed that the injected paracrine factor has similar effects with transplanted BMSCs on inhibiting astrocytes apoptosis and reactive astrogliosis after ischemic stroke. The above results indirectly confirmed that BMSCs can protect the brain injury caused by ischemic stroke by paracrine factors; meanwhile astrocytes apoptosis and reactive astrogliosis are both inhibited.Ischemic stroke is caused by blood vessel occlusion and constitutes the majority of the stroke cases. The deprivation of oxygen, glucose, and other nutrients caused by ischemic insult results in the dysfunction of neurovascular units consisting of glial cells, endothelial cells, pericytes, and neurons. The subsequent reperfusion can bring inflammation and oxidative damage, which aggravates the situation. This type of cell has several functions, such as glutamate uptake and release, homeostasis, water transportation, inflammatory response, maintenance of the blood-brain barrier and secretion of neurotrophic factors, which are all essential to normal neuronal activity and neurovascular homeostasis. Thus, it is of great significance to explore the role of astrocytes in BMSC-mediated protection against ischemic injury. To investigate whether and how paracrine factors secreted by BMSCs could regulate astrocyte survival and reactive astrogliosis after ischemic stroke, we established an in vitro ischemic stroke model (OGD), to mimic the effects of ischemic stroke and assess neurophysiological changes. Then, OGD-induced astrocytes were reperfused with BMSC-CM or co-cultured with BMSCs to mimic the effects of paracrine factors on injured astrocytes (OGD/reperfusion). To assess the molecular mechanisms, the activator and inhibitors of JNK and p38 MAPK were used to further determine whether paracrine factors secreted by BMSCs could regulate astrocyte survival and reactive astrogliosis after ischemic stroke via JNK and p38 MAPK. Meanwhile, downstream molecules, p53 and STAT1, were also investigated. The results demonstrated that paracrine factors secreted by BMSCs could protect astrocytes from OGD/reperfusion-induced injury in vitro model. Astrocyte apoptosis and GFAP upregulation of reactive astrocyte were both attenuated. The results also indicated paracrine factors could inhibit JNK and p38 MAPK, most likely through regulating the downstream targets, p53 and STAT1, to promote astrocyte survival with down-regulated GFAP after ischemic stroke in vitro.Our previous in vitro and vivo study had confirmed that BMSCs could protect astrocytes from MCAO and OGD/reperfusion-induced apoptosis and reactive astrogliosis. However, whether BMSCs could influence other functions of OGD/reperfusion-induced astrocytes remains to be investigated. As we all known, dysfunction of astrocytes led by ischemic insult can remarkably influence the generation, progress and prognosis of ischemic stroke, which can cause cell apoptosis, reactive astrogliosis, large production of ROS, releasing inflammatory cytokines, trophic factor secretion disorder, glial scar formation, water balance disorders, and glutamate metabolism. The above pathological processes aggravate the injury of nerve cell and nerve dysfunction. To investigate the paracrine effect of BMSCs on OGD/reperfusion-induced injury of astrocytes, we established an in vitro ischemic stroke model (oxygen-glucose deprivation) to mimic the effects of ischemic stroke and assess neurophysiological changes. Then OGD-induced astrocytes were reperfused with BMSC-CM or co-cultured with BMSCs to mimic the effects of paracrine factors on injured astrocytes. Then the production of ROS, the mRNA expressions of inflammatory mediators and cytokines (COX2, iNOS, TLR4, IL-6, IL-1β, TNF-α, IL-10), neurotrophic factors (VEGF, BDNF, bFGF), AQP4, glutamtae transporter (EAAT1, EAAT2) were analyzed. The results demonstrated that when OGD/reperfusion-induced astrocytes were co-cultured with BMSCs or treated with BMSC-CM, the production of ROS and expressions of COX2, iNOS, TLR4, IL-6, IL-1β, TNF-α and AQP4 were reduced, while the expressions of IL-10, EAAT1 and EAAT2 were increased. The above results confirmed that BMSCs could promote the dysfunction of OGD/reperfusion-induced astrocytes by paracrine factors.Part one:Effects of paracrine factors secreted by BMSCs on the recovery of MCAO-induced injury, and the activity and reactive astrogliosis of injured astrocytes in vivo.Objective:To investigate the effects of paracrine factors on the treatment of ischemic stroke model, and the activity and reactive astrogliosis of injured astrocytes induced by ischemic stroke in vivo.Methods:An in vivo ischemic stroke model (MACO) was established to mimic the effects of ischemic stroke and assess neurophysiological changes. At the acute of ischemic stroke, BMSCs and their paracrine factors (BMSCs supernatant) were injected to the lesions. After 7 days, cerebral infarction area and neurological recovery were assessed. Nerve Cell apoptosis was assessed by TUNEL. The protein expressions of Caspase3 and GFAP were detected by immunofluorescence and Western bolt.Results:Differences between groups were statistically significant (p< 0.05). Both transplanted BMSCs (BM group) and paracrine factors (CM group) could reduce infarct size and promote the recovery of neurological function significantly compared with the untreated group (vehicle group), (p<0.05). The benefit effects of BM group were more remarkable than CM group (p<0.05). What’s more, transplanted BMSCs (BM group) and paracrine factors (CM group) could both reduce nerve cell apoptosis and the protein expressions of Caspase3 and GFAP significantly compared with the untreated group (vehicle group), (p<0.05).Conclusions:The above results indirectly confirmed that BMSCs can protect the brain injury caused by ischemic stroke by paracrine factors; meanwhile astrocytes apoptosis and reactive astrogliosis are both inhibited.Part two:Paracrine factors secreted by BMSCs promote astrocytes survival with down-regulated GFAP after ischemic stroke by inhibiting JNK and p38 MAPK in vitroObjective:To investigate whether and how paracrine factors secreted by BMSCs could regulate astrocyte survival and reactive astrogliosis after ischemic stroke in vitro.Methods:An in vitro ischemic stroke model (OGD) was established to mimic the effects of ischemic stroke and assess neurophysiological changes. OGD-induced astrocytes were reperfused with BMSC-CM or co-cultured with BMSCs to mimic the effects of paracrine factors on injured astrocytes (OGD/reperfusion). After 24h reperfusion, the cell viability was detected by CCK8; the cell apoptosis was assessed by TUNEL; the protein expressions of apoptotic proteins, Bax, Bcl2 and Caspase3, were detected by Western blot. For the reactive astrogliosis, the immunofluorescence of GFAP was used to reflect the changes of astrocyte morphology; meanwhile the protein expression of GFAP was detected by Western blot. To assess the molecular mechanisms, the activator and inhibitors of JNK and p38 MAPK were used to further determine whether paracrine factors secreted by BMSCs could regulate astrocyte survival and reactive astrogliosis after ischemic stroke via JNK and p38 MAPK. Meanwhile, downstream molecules, p53 and STAT1, were also investigated. After the above treatments, the injured astrocytes were detected by the aforementioned methods.Results:Differences between groups were statistically significant (p< 0.05). Both co-culturing with BMSCs (co-cultured group) and reperfusion with paracrine factors (conditioned group) could increase the cell viability of OGD/reperfusion-induced astrocytes (p<0.05); reduce the apoptosis (p<0.05); significantly downregulated bax and caspase3 protein expression, and upregulated the protein expression of bcl2 compared with the just OGD/reperfusion group (standard group) (p<0.05). Meanwhile, reperfusion of OGD-induced astrocytes with CM or co-culturing with BMSCs for 24 h reduced the number of hypertrophic cells and decreased GFAP protein expression remarkably compared with standard group (p<0.05). Western blot analysis indicated that 6 h OGD or OGD with 24 h reperfusion in the standard medium significantly upregulated the protein expressions of p-p38 and p-JNK compared with the control (p<0.05). OGD with 24 h reperfusion in the standard medium resulted in more significant upregulations of p-p38 and p-JNK than OGD for 6h (p<0.05). Nevertheless, after 24 h reperfusion with CM or co-culturing with BMSCs, paracrine factors reduced p-p38 and p-JNK protein expression dramatically compared with the standard group (p<0.05). The results also showed that after 24 h reperfusion with CM or co-culturing with BMSCs, paracrine factors reduced p-p53, p53 and p-STAT1 protein expression dramatically compared with the standard group (p<0.05). TUNEL assay showed that the apoptotic rate significantly decreased in the inhibitors of p38 MAPK and JNK treated groups compared with the standard group, and the CCK8 assay indicated that cell viability increased remarkably in both groups (p<0.05). Western blot analysis of pro-apoptotic (bax and caspase 3) and anti-apoptotic (bcl2) proteins showed that the treatment with p38 MAPK or JNK inhibitor significantly downregulated the protein expressions of bax and caspase 3, and upregulated bcl2 protein expression compared with the standard group (p<0.05). Meanwhile, inhibition of p38 MAPK and JNK significantly decreased protein expression of GFAP compared with the standard group (p<0.05). For the p38 MAPK and JNK activator, TUNEL assay showed that the apoptotic rate significantly increased in the anisomycin group compared with the standard group, and the CCK8 assay indicated that cell viability in the anisomycin group was reduced remarkably as well (p<0.05). In addition, the activator of p38 MAPK and JNK significantly upregulated bax and caspase3 protein expression, and downregulated the protein expression of bcl2 compared with the standard group (p<0.05). Once anisomycin was added to the CM (reperfusion for 24 h, CM/anisomycin group), the beneficial effects of paracrine factors on astrocyte survival were suppressed. The apoptotic rate, bax and caspase3 protein expression increased, and cell viability and bcl2 protein expression were reduced notably compared with the CM group (p<0.05). The activation of p38 MAPK and JNK significantly upregulated the protein expression of GFAP compared with the standard group (p<0.05). In addition, once anisomycin was added to the CM (reperfusion for 24 h, CM/anisomycin group), the down-regulated effects of paracrine factors on GFAP expression was suppressed (p< 0.05).For the inhibition of p53 and STAT1, CCK8 assay indicated that when OGD-induced astrocytes were reperfused with PFT-α, Flu or PFT-α/Flu for 24 h, cell viability improved in the three groups compared with the standard group (p< 0.05). To further investigate the apoptosis of OGD/reperfusion-induced astrocytes, bax, caspase3, and be 12 were detected by western blot. PFT-α, Flu and PFT-α/Flu downregulated bax and caspase3 protein expression, and upregulated the protein expression of bcl2 remarkably compared with the standard group (p< 0.05). To further investigate whether p53 and STAT1 participate in regulating GFAP expression induced by OGD/reperfusion, GFAP expression was detected by western blot. GFAP protein expression was decreased by Flu and PFT-α/Flu (p< 0.05), but not PFT-α (p> 0.05) compared with the standard group.Conclusions:In conclusion, paracrine factors secreted by BMSCs protect astrocytes from OGD/reperfusion-induced injury, attenuating apoptosis and GFAP overexpression via suppressing the activation of p38 MAPK and JNK, most likely by regulating their downstream targets, p53 and STAT1, after ischemic stroke in vitro.Part three:Effects of paracrine factors secreted by BMSCs on the other functions of OGD/reperfusion-induced injured astrocytes in vitro.Objective:To investigate the effects of paracrine factors on the oxidative stress, inflammation, neurotrophic factors secretion, AQP4 and glutamate transporter expressions of OGD/reperfusion-induced injured astrocytes in vitro.Methods:An in vitro ischemic stroke model (oxygen-glucose deprivation) was established to mimic the effects of ischemic stroke and assess neurophysiological changes. Then OGD-induced astrocytes were reperfused with BMSC-CM or co-cultured with BMSCs to mimic the effects of paracrine factors on injured astrocytes. Then the production of ROS was assessed by DCFH-DA; the mRNA expressions of inflammatory mediators and cytokines (COX2, iNOS, TLR4, IL-6, IL-1β, TNF-α, IL-10), neurotrophic factors (VEGF, BDNF, bFGF), AQP4, glutamtae transporter (EAAT1, EAAT2) were detected by qPCR.Results:Differences between groups were statistically significant (p<0.05). When OGD/reperfusion-induced astrocytes were co-cultured with BMSCs or treated with BMSC-CM, the production of ROS and expressions of COX2, iNOS, TLR4, IL-6, IL-1β, TNF-α and AQP4 were reduced, while the expressions of IL-10, EAAT1 and EAAT2 were increased with the standard group (p< 0.05).Conclusions:Paracrine factors secreted by BMSCs could reduce the production of ROS and expressions of COX2, iNOS, TLR4, IL-6, IL-1β, TNF-α and AQP4; increase the expressions of IL-10, EAAT1 and EAAT2 to promote the dysfunction of OGD/reperfusion-induced astrocytes. |
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