| Cerebral ischemia-reperfusion injury is an extremely complicated pathophysiologic process, involves a lot of kinds of mechanisms, such as the production of free radicals, excitotoxicity, intracellular calcium overloading and so on, and each other form a complex network. Although the study of mechanism of cerebral ischemia-reperfusion injury in the deepening and development, but the brain ischemia reperfusion injury mechanisms intersection is unclear, hoping to a more in-depth study. Explore a complex series of pathophysiological events that evolve in time of cerebral ischemia-reperfusion injury and explore STV effect on the protection of ischemia reperfusion injury in rats and its protection mechanism.We establish middle cerebral artery occlusion and reperfusion(MCAO/R)model considering the method of Longa.Using Laser Doppler flowmeter to implement real-time detection and analysing the value of blood flow changes, to ensure the reliability of the model building. Real-time monitoring in the rat heart rate, breath rate, blood oxygen saturation, and and temperature, to exclude other factors on the impact of animal model building. Ethology score on each group of rats. Application of TTC staining to judge the success of cerebral infarction, at the same time using Image J software to calculate the infarction area, to measure STV effect on the cerebral stroke. To explore a reasonable and effective separation system for brain tissue protein, we use different concentrations(10% T, 2.6% C or 12% T, 2.6% C or 15% T, 2.6% C) SDS-polypropyleneamide gel and 4.2%-17.85% T, 5% C linear gradient SDS-polypropyleneamide gel for rat brain protein separation. Using Western blot technology to detect Thioredoxin 1, Peroxiredoxin 2 protein and Heat shock protein 60 kDa expression respectively in each group of corresponding time ischemia 2 hours /reperfusion 0 hour/6 hours/22 hours/168 hours, to explore whether STV have nerve protective effect and its possible mechanism. For the research, we successfully build the ischemia/reperfusion rats model, application TTC staining to evaluate cerebral infarction area, found STV group and positive group rats than ischemia grop has less cerebral infarction area, at the same time for ischemia 2 hours /reperfusion 22 hours this point model, STV group rats than positive control rat has larger cerebral infarction area, but for ischemia 2 hours /reperfusion 168 hours model, STV group rats than positive group rat has less cerebral infarction area. To some extent, STV has certain protective effect on cerebral ischemia and its long-term protective effect is more apparent, stronger than the positive drug. We test several different concentrations(10% T, 2.6% C or 12% T, 2.6% C or 15% T, 2.6% C) SDS-polypropyleneamide gel and 4.2%-17.85% T, 5% C linear gradient SDS- polypropyleneamide gel system, eventually found 4.2%-17.85% T, 5% C linear gradient SDS-polypropyleneamide had good separation effect for rat brain tissue protein.The results of western blotting show that ischemia 2 hours/reperfusion 0 hour and 6 hours model, STV group decreased Hsp60 expression, this explain STV in early time can reduce stress reaction in brain tissue. In ischemia 2 hours /reperfusion 22 hours, Prx2 in STV group was obviously higher and in ischemia 2 hours /reperfusion 168 hours model, the expression of Trx1 in STV was higher than the positive group. Explain Prx2 plays a good antioxidant effect in cerebral ischemia, and prevent free radicals damage to brain tissue. Trx1 play a good role of oxidation resistance and resistance to apoptosis in cerebral ischemia, and prevent the death of neurons. For ischemia 2 hours/reperfusion 22 hours, in STV group Prx2 expression quantity is higher than Trx1 expression at the same period, explaining for ischemia 2 hours /reperfusion 22 hours model, STV mainly play a role of antioxidant through raising Prx2 expression. For ischemia 2 hours /reperfusion 168 hours, STV mainly play a role of resistance to apoptosis through raising Trx1 expression. Experimental results show that STV start different signaling pathways to protect the brain tissue at different stage of cerebral ischemia.Except the work we explain above, we still do another job. The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting and quantitative LC-MS/MS(in data-independent acquisition mode, or MS~E), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS~E(HDMS~E)and applied to analyze the area of human plasma low-density lipoprotein(LDL)An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gelpieces and subjected to quantitative LC-MS/MS. Compared with MS~E, HDMS~E showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. Totally 253 proteins were assigned with LC-HDMS~E and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentratedat p I ca.5.4-6.1 and apparent mass ca.1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which show protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which nineteen proteins have been reported to be associated with LDL. This method requires only several microliters of a plasmasample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL. |
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