| As a populous country, China has become an aging society. Age is a not modifiable risk factor for cerebrovascular disease. Stroke prevalence increased gradually with the increase of age. The prevalence of stroke reached the peak at 60 ~ 64 years old, then gradually decreased. The data from China stroke conference in 2014 showed that the prevalence of stroke increased with the rate of 8.1% per year in China in recent 25 years. Cerebral infarction is one of the most common types of stroke, accounting for 78% of all stroke patients. The mechanism of the occurrence and development in ischemic stroke is very complicated and not completely clear. Therefore, in recent years, the exploration of the mechanism of the occurrence and development in ischemic stroke has become a focus and difficulty of the research in the field of neuroscience.Autophagy is a lysosome mediated intracellular metabolic mechanism. The mechanism is responsible for the degradation and recycling of cellular components and damaged organelles which have lost function. The main process of autophagy includ the formation of autophagosome and autolysosome.The autolysosome is a fusion of autophagosome and lysosome. In recent years, a growing number of studies suggested that autophagy is involved in cerebral vascular disease. But there is still no unified conclusion about the role of autophagy in cerebral vascular disease. Cerebral ischemia can activate autophagy through a variety of signaling pathways. This study mainly discusses the role of class ? PI3K-Beclin 1 signaling pathway in ischemic cerebrovascular disease.First, mouse neuroblastoma cells( N2a) exposed to oxygen and glucose deprivation( OGD) were used to simulate cerebral ischemia injury. Autophagy related proteins include microtubule-associated protein 1 light chain 3( LC3), Beclin 1, P62(also known as Sequestosome 1, SQSTM1), lysosome-associated membrane protein 2(LAMP2)and so on. Western blot analysis was applied to detecte the expression of these proteins. Acid phosphatase activity and cell viability of N2 a cells which had undergone OGD treatment also were determined. Then, N2 a cells were administrated with 3-methyl adenine(3-MA), which is the PI3 K inhibitor. The expression of the autophagy-related poteins, acid phosphatase activity and cell viability were determined after 3-MA administrated during OGD treatment. Finally, shRNA lentiviral technology was applied to interfere the expression of Beclin 1 gene in N2 a cells. The autophagy-related protein expression, acid phosphatase activity and cell viability also were detected after OGD treatment in N2 a cells, in which Beclin 1 was knocked down.The results showed that: 1) The level of LC3 ?, Beclin 1, p62/ SQSTM1 and LC3 ?/? ratio in N2 a cells were markedly increased, but the level of LAMP2 did not significantly change after OGD treatment; OGD treatment showed an enhancement in the acid phosphatase activity and a decrement in cell viability in N2 a cells. 2) 3-MA(5mM) treatment reduced the level of LC3 ? and LC3 ?/? ratio, increased the expression of p62 /SQSTM1 protein and had little effect on the level of Beclin 1 and LAMP2 during OGD treatment; After 3-MA treatment, acid phosphatase activity and cell viability in N2 a cells were significantly decreased. 3) When Beclin 1 was knocked down by shRNA, the level of LC3 ?, p62/ SQSTM1 and LC3 ? / ? ratio were markedly increased, but the level of LAMP2 did not change significantly in N2 a cells. The acid phosphatase activity also was significantly increased, but the cell viability had no obvious change in N2 a cells, in which Beclin 1 gene has been interfered.In summary, the present study demonstrate that: 1) OGD treatment induces Beclin 1-independent autophagy in N2 a cells, and this kind of autophagy is not regulated by class ? PI3K-Beclin 1 signaling pathway; 2) The increased autophagic activity during OGD insult play a protective role; 3) The expression level of p62/SQSTM1 does not always inversely correlate with autophagy activity. |
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