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Relationship Between Protect Effect Of Tong-Qiao-Huo-Xue-Decoction On Cerebral Ischemia Reperfusion Injuryand Autophagy Via The Autophagy Signaling Pathway

Posted on:2017-07-10Degree:MasterType:Thesis
Country:ChinaCandidate:G Y WangFull Text:PDF
GTID:2334330485956522Subject:Pharmacy
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Objective:A model of ischemic stroke in the rat was established after transient middle cerebral artery occlusion(MCAO) followed by reperfusion. Here we investigated how TQHXD influences autophagy, a cellular damage occurring during the ischemia reperfusion injury and its relevant mechanism.Methods:1. Preliminary study on the autophagy after cerebral ischemia reperfusion in rats(1) Western blot analyses showed the level of Beclin 1 and LC3 in the mice model of ischemia-reperfusion.(2) Electron micrographs of morphological changes of brain tissues following ischemia-reperfusion injury.(3) Nissl's staining in the mice model of ischemia-reperfusion.(4) Cellular distribution of LC3 in rat brain after middle cerebral artery occlusion by double immunostaining2. Neuroprotective effect of TQHXD after cerebral ischemia reperfusion We used neurological deficits and infarct volume to explore the protective effect of TQHXD after cerebral ischemia reperfusion.3. Effects of TQHXD on the expression of autophagy after cerebral ischemia reperfusion(1) Rats were treated with TQHXD(6g/kg), the neurological deficit, the cerebral infarct size and HE stainning were examined, and the levels of relevant proteins were determined by immunoblotting analysis and immunofluorescent staining.(2) Rats were treated with TQHXD and 3-MA, the neurological deficit, the cerebral infarct size and HE stainning were examined, and the levels of relevant proteins were determined by immunoblotting analysis and immunofluorescent staining.(3) Rats were treated with TQHXD and Rapa, the neurological deficit, the cerebral infarct size and HE stainning were examined, and the levels of relevant proteins weredetermined by immunoblotting analysis and immunofluorescent staining.4. Relevant mechanism of TQHXD regulating autophagy after cerebral ischemia reperfusion Western blotting analysis was determined to detect the effects of TQHXD on the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(m TOR) signaling pathway.Results1. Preliminary study on the autophagy after cerebral ischemia reperfusion in rats(1) Ischemia reperfusion elevated the levels of microtubule-associated protein1 light chain 3(LC3) and Beclin-1.(2) We could observe the autophagosome by transmission electron microscope when ischemia 2 h reperfusion 24 h.(3) Nissl staining showed that ischemia 2 h reperfusion 24 h has the most serious damage.(4) Double immunostaining showed that cell distribution of LC3 can be observed in neurons?astrocytes and brain microvascular endothelial cells.2. Neuroprotective effect of TQHXD after cerebral ischemia reperfusion TQHXD could improve the neurological deficits and reduced the infarct volume.3. Effects of TQHXD on the expression of autophagy after cerebral ischemia reperfusion(1) Rats treated with TQHXD(6g/kg) could significantly improve the neurological deficit and HE stainning, reduce the cerebral infarct size, inhibit the levels of Beclin-1and LC3 compared to the model group.(2) Rats treated with TQHXD and 3-MA could also significantly improve the neurological deficit and HE stainning, reduce the cerebral infarct size, inhibit the levels of Beclin-1 and LC3 compared to the model group.(3) Rats treated with TQHXD and Rapa could not significantly change the neurological deficit?HE stainning?infarct size, the levels of Beclin-1 and LC3 compared to themodel group4. Relevant mechanism of TQHXD regulating autophagy after cerebral ischemia reperfusion TQHXD could up-regulate the level of p-AKt and p-m TOR, while inhibit the expression of Beclin-1 and LC3.Conclusion Ischemia reperfusion elevated the levels of autophagy related proteins(LC3 and Beclin-1) and could observe the autophagosome by transmission electron microscope.Double immunostaining showed that cell distribution of LC3 can be observed in neurons ? astrocytes and brain microvascular endothelial cells. TQHXD could up-regulate the PI3K/Akt/m TOR signaling pathway, inhibiting the expression of Beclin-1 and LC3 to exert a protective effect.
Keywords/Search Tags:Tong-Qiao-Huo-Xue-Decotion(TQHXD), Autophagy, Ischemia reperfusion, phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)(PI3K/Akt/mTOR)
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