| BackgroundIschemic cerebrovascular disease, with high mortality and disability rate, is the most common disease among aged people. Many of survivors tend to suffer from different degrees of dysfunction. these people are heavy burden of family and society.Thrombolytic therapy is the preferred treatment for cerebral infarction, but the majority of patients can't benefit from it due to time window and the limitation of technical conditions.There are no effective therapy for cerebral infarction expect thrombolytic therapy. A lot of study on stem cell transplant therapy of ischemic brain injury has confirmed that stem cells can reduce ischemic brain damage,but the specific mechanism is not entirely clear.PI3K/ Akt is a classic antiapoptotic pathway, which is closely connected with ischemic brain damage. Studies have shown that the activation of this pathway can reduce brain injury after ischemia. However, it has not been reported yet whether human umbilical cord blood mesenchymal stem cells can activate this pathway, regulate inflammatory reaction, inhibit of nerve cell apoptosis, protect nerve cells through the pathways after ischemia.In this research, after umbilical cord blood mesenchymal stem cell transplantation in rat middle cerebral artery occlusion (MCAO) model, we observed serum inflammatory factor, the Bcl-2, change of Bax, nerve cells apoptosis, PI3K/Akt signaling pathways expression and neural function score. Based on the observation, we explored: whether umbilical cord blood mesenchymal stem cells can adjust PI3K/Akt signaling pathways, so as to reduce inflammatory reaction caused by ischemic brain injury, inhibit cell apoptosis, and improve neural function in rats.Part 1: Nerve protective effect of human umbilical cord blood mesenchymal stem cells transplantation on rats with ischemic brain injuryObjectiveThis part of test was designed to observe effect of human umbilical cord blood mesenchymal stem cell transplantation on inflammatory factor regulation, nerve function and cell apoptosis in rats with ischemic brain injury.MethodsWe selected clean and healthy adult male SD rats to prepare left middle cerebral artery occlusion model using improved suture method. The rats were randomly divided into sham group, model group, Normal saline group , HUCB-MSCs group, with 15 rats in each group. Experimental period lasted 2 weeks.Sham group: Operative procedure was the same with model group, but without endovascular filament for occlusion.Model group: Middle cerebral artery occlusion model was prepared, without any treatment.Normal saline group: 24 hours after the preparation of middle cerebral artery occlusion, 1 ml saline solution was injected via left femoral venous.HUCB-MSCs group: 24 hours after the preparation of middle cerebral artery occlusion, 1 ml sixth generation umbilical cord blood stem cells (3 x 106 cells) saline dilution was injected via left femoral venous.All rats were taken blood via tail vein and assessed with neurological scores using mNSS methods before ischemia. After ischemia, rats were taken blood at time points of 24hours,3 days,7 days, and 14 days. Meanwhile, mNSS score was used to evaluate neurologic impairment. Then, rats were decapitatedto observe nerve pathological morphological changes using HE staining, and cell apoptosis by in situ apoptosis(TUNEL) detection.Results1. Neurological deficit scores:For rats in Sham group, no obvious change was found at each time point. The mNSS scores of Model group, Normal saline group , HUCB-MSCs group at each time point after ischemia were significantly higher than that of Sham group (P < 0.05).Concerning neurological scores, there was no significant differences among Model group, Normal saline group and HUCB-MSCs group 24 hours after ischemia; while it was obviously lower in HUCB-MSCs group than Model group and Normal saline group at 3 days, 7 days and 14 days after ischemia (P < 0.05).2. Inflammatory factor levelsThere were no significant changes at each time point in Sham group. Serum IL-10 and TGF-?1 of Model group and Normal saline group decreased significantly after ischemia, with the lowest level of IL-10 at time point of 24 hours after ischemia and the lowest TGF-?1 level 3 days after cerebral ischemia. Treated by stem cell transplantation,serum IL-10 level increased significantly with the highest value 3 days after ischemia,followed by a gradual decline, which was higher than that of Sham group (P<0.05) at 7 days after ischemia, and returned to normal level 14 days after ischemia. TGF-?1 of HUCB-MSCs group was significantly higher than that of Model group, Normal saline group and Sham group (P<0.05) 3 days after ischemia, which then declined rapidly,slightly lower than that of Sham group, but significantly higher than that of Model group and Normal saline group (P<0.05). Concerning IL-6 and IFN-? levels, no obvious changes were detected at each time point; while the two levels in other groups significantly increased after ischemia. The values reached the maximum 24 hours later,then dropped slowly,but did not return to normal levels 14 days later,which was still significantly higher than the Sham group (P<0.05). In comparison, IL-6 and IFN-?levels in HUCB-MSCs group were significantly lower than that of Model group and Normal saline group (P<0.05) .3 days, 7 days and 14 days after ischemia; while no obvious difference was found compared with Sham group.3. TUNEL stainingThere were no obvious pathological changes of cell mor:phology at each time point in Sham group. In Model group and Normal saline group, necrosis was found around infarction, where with many visible hyperchromatic, pyknotic nuclei. Similar pathological changes appeared in HUCB-MSCs group;but necrosis and apoptosis were superior to the two groups, with TUNEL positive cells significantly lower (p < 0.05).ConclusionThe therapy of human umbilical cord blood mesenchymal stem cell transplantation at early stage in ischemic brain injury is helpful to regulate inflammatory reaction,reduce apoptosis, improve nerve function score, and thus promote neural functional recovery.Part 2: Study of human umbilical cord blood mesenchymal stem cells modulating inflammatory response and inhibiting neuron apoptosis through PI3K/Akt signaling pathways in rats with ischemic brain injuryObjectiveThis part of test was designed to observe effect of human umbilical cord blood mesenchymal stem cells transplantation on, inflammatory response adjustment and apoptosis inhibition via PI3K/AKt signaling pathways in rats with ischemic brain injury.MethodsClean and healthy adult male SD rats were randomly divided into sham group,model group, HUCB-MSCs plusLY294002 group, and HUCB-MSCs group, with 15 rats in each group. Experimental period lasted 2 weeks. Left middle cerebral artery occlusion model was prepared using improved suture method.Sham group: Operative procedure was the same with model group, but without endovascular filament for occlusion.Model group: Middle cerebral artery occlusion model was prepared, without any treatment.HUCB-MSCs plusLY294002 group: 24 hours after the preparation of middle cerebral artery occlusion, 0.1 ml LY294002 diluent andl ml sixth generation umbilical cord blood stem cells (3 x 106cells ) saline dilution was injected via left femoral venous.HUCB-MSCs group: 24 hours after the preparation of middle .cerebral artery occlusion,1 ml sixth generation umbilical cord blood stem cells (3 x 106cells) saline dilution wasinjected via left femoral venous.All rats were taken blood via tail vein and assessed with neurological scores using mNSS methods before ischemia. After ischemia, rats were taken blood at time points of 24hours,3 days, 7 days, and 14 days. Meanwhile, mNSS was used to evaluate neurologic impairment. Then, rats were decapitated to test Akt and P-Akt expression of brain tissues using Western blot method, and cell apoptosis of brain tissues by in situ apoptosis (TUNEL) detection.Results1. Neurological deficit scores:For rats in Sham group, no obvious change was found at each time point.Psychomotor function of Model group, HUCB-MSCs plusLY294002 group, and HUCB-MSCs group at each time point after ischemia were significantly higher than that of Sham group. There was no significant differences among Model group,HUCB-MSCs plusLY294002 group and HUCB-MSCs group before transplantion (24 hours after ischemia). but after transplantion (3 days, 7 days, and 14 days after ischemia), score of Model group remained high as the highest in the four groups. Score of HUCB-MSCs plus LY294002 group decreased with significantly been lower than Model group (P < 0.05)and score of HUCB-MSCs group was significantly lower than HUCB-MSCs plus LY294002 group (P < 0.05).2. Inflammatory factor levelsThere were no significant changes at each time point in Sham group. Serum IL-10 and TGF-?1 decreased significantly ,while IL-6 and IFN-y levels increased in Model group after ischemia.with values After transplantion (3 days, 7 days, and 14 days after ischemia),IL-10 and TGF-?1 of HUCB-MSCs group increased obviously,significantly higher than those of Model group and HUCB-MSCs plusLY294002 group(P<0.05). While the levels of IL-10 and TGF-?1 of HUCB-MSCs plus LY294002 group observably higher than Model group(P<0.05).IL-6 and IFN-y levels obviously declined after transplantion (3 days, 7 days, and 14 days after ischemia). More significant reduction can be found in HUCB-MSCs group.IL-6 group and IFN-? levels of HUCB-MSCs group were lower than Model group and HUCB-MSCs plus LY294002 group (P<0.05).While IL-6 and IFN-y levels of HUCB-MSCs plusLY294002 group were clearly lower than Model group(P<0.05).3. p-Akt and AktExpression of p-Akt protein in Model group decreased significantly compared with Sham group, with statistically significant (P<0.05). While it increased obviously in HUCB-MSCs group and HUCB-MSCs plus LY294002 group. Expression level of p-Akt protein in HUCB-MSCs plus LY294002 group was significantly lower than HUCB-MSCs group (P<0.05), but significantly higher than that of Model group(P<0.05). No significant differences of total Akt protein were detected among the four groups.4. Bcl-2 and BaxBcl-2 and Bax in Model group raised, with Bax increasing significantly compared to Sham group (P<0.05). Bcl-2 of HUCB-MSCs plus LY294002 group increased obviously compared with Model group(P<0.05),while Bax significantly lower than that of Model group(P<0.05).Bcl-2 of HUCB-MSCs group increased obviously compared with HUCB-MSCs plus LY294002 group (P<0.05).while Bax decreased to the normal level, significantly lower than that of HUCB-MSCs plus LY294002 group (P<0.05), but with no significant difference compared with Sham group.5. TUNEL stainingThere were no obvious pathological changes of cell morphology at each time point in Sham group, with no TUNEL positive cells.While apoptosis showed in different degrees in the remaining three groups. In Model group and HUCB-MSCs plusLY294002 group, necrosis was found around infarction, with many visible more hyperchromatic,pyknotic nuclei. Apoptosis in HUCB-MSCs group was the lightest,superior to the above mentioned two groups (p<0.05).Apoptosis of HUCB-MSCs plusLY294002 group was between HUCB-MSCs group and Model group, with significantly more TUNEL positive cells compared to HUCB-MSCs group (p<0.05).ConclusionHuman umbilical cord blood mesenchymal stem cells can regulate inflammatory reaction, promote the expression of Bcl - 2, reduce the expression of Bax, inhibitcell apoptosis, improve the function of nerve, reduce ischemic brain damage through the PI3K/AKt signaling pathways after ischemic brain injury in rats. |
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