The Protective Effects Of Baicalin Postconditioning On Cerebral Ischemic/ Reperfusion Injury In Rats Via Akt-Autophagy Pathway

Objective:To study the protective effects of baicalin postconditioning on the induction of autophagy in rats model of cerebral ischemic /reperfusion injury, and further explore the potential regulation signaling pathways of autophagy.Methods:SPF male SD rats were selected for research object and rat middle cerebral artery occlusion (MCAO) was used to construct cerebral ischemic/reperfusion injury(CI/RI) model. Neurological scores and Nissl stainning were used to observe the neurons injury; Immunohistochemistry was used to detect the expression of microtubule associated protein light chain 3 (LC3); The expression of AKT、Beclinl and LC3 after treatement with baicalin were detected by Western blot and Real-time QPCR respectively; Apoptosis related factor caspase-3 protein expression was tested by Western blot and the volume of brain infarct was evaluated by TTC staining after application of autophagy inhibitor 3-methyl adenine(3-MA); furthermore,the activation of autophagy induced by Baicalin postconditioning were also tested by Western blot with LY294002, the inhibitor of AKT.Results:1. The effect of baicalin postconditioning on CI/RI. Neurological scores and Nissl stainning reflect the pathological damage of cerebral ischemia from the angle of the function and organization. Compared with I/R group, Neurological score was reduced(P<0.05) and the densitiy of Nissl body was increased in I/R+Bai group. This indicated that baicalin postconditioning alleviated the pathological injury of cerebral ischemia.2. The effect of baicalin postconditioning on autophagy after CI/RI. Immunohistochemistry results showed:Expression of LC3 in Sham group maintained at a low level,while in I/R group showed a higher level,furthermore showed a strong expression in I/R+Bai group. Western blot results showed:Compared with Sham group, LC3-II/LC3-I ratio and Beclinl expression level were increased in I/R group, respectively (P<0.01),furthermore, a greater increase was observed in I/R+Bai group (P<0.01). Real-time QPCR results showed:Compared with Sham group, LC3 and Beclinl mRAN transcriptional level were increased in I/R group, respectively (P<0.05); furthermore, a greater increase with LC3 mRNA(P<0.05)and also Beclinl mRAN (P<0.01)was observed in I/R+Bai group. These results indicated that baicalin postconditioning could father induce the activation of autophagy after CI/RI.3. The role of autophagy in baicalin postconditioning against CI/RI. TTC and Western blot results showed:compared with I/R group, LC3-Ⅱ/LC3-Ⅰ ratio was raised, cerebral infarction volume and the protein expression level of apoptosis related gene caspase-3 were reduced in I/R+Bai group, respectively(P<0.01); while after application of autophagy inhibitor 3-MA, LC3-Ⅱ/LC3-Ⅰ ratio was reduced, but cerebral infarction volume and caspase-3 protein expression level were increased (P<0.05). These results indicated that application of autophagy inhibitor could eliminate the protective effect of baicalin postconditioning on CI/RI. In other words, autophagy induction contributes to the neuroprotection of baicalin postconditioning against CI/RI and was negatively correlated with apoptosis induction.4. The effect of baicalin postconditioning on AKT expression after CI/RI. Western blot and Real-time QPCR results showed:compared with I/R group, AKT phosphorylation protein expression level and mRNA transcriptional level were increased in I/R+Bai group (P<0.01), indicating AKT pathway was actived by baicalin postconditioning after CI/RI.5. The effect of baicalin postconditioning on autophagy after application of AKT inhibitor LY294002. Western blot results showed: compared with I/R+Bai group, the protein level of p-AKT was reduced,and the ratio of LC3-Ⅱ/LC3-Ⅰ was also reduced (P<0.01). The results showed that LY294002 could block autophagy induced by baicalin postconditioning after CI/RI. And also revealed that induction of autophagy in baicalin postconditioning against CI/RI may be through AKT signaling pathway.Conclusion:In our study,we confirmed that the neuroprotection of baicalin postconditioning on cerebral ischemic/reperfusion injury maybe through the induction of autophagy.Furthermore, the induction of autophagy maybe mediated by the activation of AKT signaling pathway.