| Brain vascular disease is the third leading cause of death in the world, following cardiovascular diseases and cancers. The incidence and death disability of ischemic stroke is quite high. In Traditional Chinese medicine, vessel injury is the one of reasean for stroke. Treatments of ischemic stroke based on "vessel" theory is promising. Our project is to study if TLJN plays a protective role in ischemia-reperfusion injury and whether IGF-1 is evolved. We also investigate if IGF-1 plays a protective role in neurons. If so, through which signaling pathway.Objective:Investigating the protective role of TLJN in ischemia-reperfusion injury and its potential signaling pathway. Studying expression pattern of IGF-1 and IGF-1 receptor in rats and the interaction between the expression and the TLJN. In vitro study investigates the protective role of IGF-1 to the neurons and illuminate the signaling pathway. This study provides the molecular-biology foundation for the Chinese medicine "vessel theory"Methods:1. To observe the morphological changes of the rat ischemic-reperfusion brain and the influence of TLJN injection, we use TTC staining, HE staining, and immunofluorescence techniques to study the brain injuring and the protection of TLJN.2. Investigate the concentration difference of IGF-1 among different groups using Elisa; Elisa, immunohistochemistry and western blotting were used to detect the expression of IGF-1 and IGF-1R of ischemic brain, and observe the difference of the expression when TLJN injection was used or not.3. New born rat cerebral cortical neurons were cultured in vitro, at day 7, neurons were divided into 8 groups, four groups treated with IGF-1 and (or) LY294002 (PD98059) were exposed to OGD. MTT assay was used to analyze the viability of neurons in each group; The expression of total Akt and p-Akt were analyzed by western blotting.4. Investigate the expression of p-Akt and p-p44/42MAPK of different groups, which are the most marker proteins of PI3K and MAPK pathway. Then to infer the possible growth Results:1. By comparing the neurobehavioral effect of TLJN treated group, positive control group (treated by edaravone), and negative control group, we found that TLJN group shows significant difference comparing with other groups. Although all three groups exists brain infarction, the infarction area of TLJN group is much smaller that the negative control group. Comparing with positive control group, similar effects were found and there is no statistically significance between the TJLN group and positive control group. The sham operation group shows neurons of brain cortex and hippocampus are well organized; neuron fibers are intact with high MAP-2 fluorescence signal. Similar results were found on the untreated side of all three groups. We found significant decrease of MAP-2 expression level in the negative control group, especially in the infarct area with extensive fluorescent dark zone. Comparing to the negative control group, the intensity of MAP-2 fluorescence of TLJN is higher and the neuron fibers are relatively complete. We didn't find significant change of MAP-2 expression in the hippocampus area of the brain in both TLJN group and negative control group.2. ELISA test of serum IGF-1 results show that negative control group has dramatically decreased IGF-1 level 24 hours after ischemia treatment while the TLJN has significant increased IGF-1 level 3 days after ischemia treatment. There are no differences between the three time points of sham groups. Immunohistochemistry study shows that comparing to the sham operation group, the percentage of IGF-1 expression positive area is higher in 24 hours and 3 days time points of negative control group while there is no difference in 7 days time points. The TLJN group has higher percentage of IGF-1 positive area at all three time points comparing to other groups.3. IGF-1R immunohistochemistry of ipsilateral cortex showed that there is no significant difference of the positive expression of IGF-1R area between the three time points in the sham operation group. Comparing with the sham group, at 24-hour time points, IGF-1R positive area was significantly increased in TLJN group; Similarly, the group's IGF-1R expression were significantly higher than the same time points from other group. By comparing the 3 and 7 day time points, we didn't find dramatic difference between all three groups yet there is small increase of TLJN expression level (no statistically significance). These data were confirmed by western blotting technique by testing IGF-1R protein levels in rat ischemia brain tissues.4. Compared with the control, the neuron viability was significantly higher in IGF-1 treated group under norma or OGD condition (P<0.05). The protective effects of IGF-1 were attenuated in the presence of LY294002 but not PD98059. The result of western blotting showed IGF-1 upregulated the expression of pAkt, which was inhibited by Ly294002.5. At day 3, the expression of p-Akt and p-p44/42MAPK in model group reduced significantly compared with the sham group; while the expression was upregulated in the TLJN group.Conclusions:1. TLJN injection has protective effect on rat brain ischemic-reperfusion injury by improving the neurologic function, decreasing the infarction volumn, which maybe related to the upregulated expression of IGF-1 and IGF-1R of the ischemic brain.2. IGF-1 has important neuroprotective effects. Compared with the control, the neuron viability was significantly higher in IGF-1 treated group under normal or OGD condition. PI3K pathway may play an important role in neuroprotection afforded by IGF-1.3. PI-3K and MAPK pathway may be activated by the use of TLJN. Then growth factors which can be expressed in the brain or can pass through the blood-brain barrier, and may activate PI-3K and (or) MAPK. pathway, are be inferd as the possible factors secreated in by the use of TLJN, all of which are to be explored further. |
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