The Influences And Mechanisms Of Calycosin Against Focal Cerebral Ischemia And Reperfusion Injury In Rats

Objective: Calycosin is one of the main active components of Astragalus membranaceus in traditional Chinese medicine,and has obvious characteristics of phytoestrogen.In previous studies,it has shown some certain effects respectively in anti-virus and the inhibition of human malignant tumor proliferation,especially in cardiovascular diseases,which attracted much attention in recent years.It has been reported that estrogens could provide neuroprotective effects,and dietary intake of phytoestrogens could reduce stroke injury in cerebral ischemia/reperfusion(I/R)animal models.Emerging evidence suggests that autophagy plays important roles in the pathophysiological processes of cerebral ischemia and reperfusion injury.P62(also known as sequestosome-1/SQSTM1)is a selective substrate of autophagy and a multidomain scaffold protein.Similar to p62,NBR1 also can mediate the degradation of ubiquitinated cargos through their interaction with ubiquitin.Recent studies showed that there exists an intricate crosstalk among apoptosis,inflammatory and autophagy.In our previous studies,we concluded that neuroprotective mechanisms of calycosin against focal cerebral ischemia and reperfusion injury in rats,but mechanism is not clear.In our studies,We construct the middle cerebral artery occlusion in male rats(middle cerebral artery occlusion,MCAO).Research the influences and mechanisms of calycosin from the molecular,morphological and animal levels aspects in cerebral ischemia reperfusion injury on brain neurons and related of autophagy,apoptosis and inflammation factors.Methods:(1)Middle cerebral artery occlusion(MCAO)model was established in adult male Sprague-Dawley rats,simulating of cerebral ischemia-reperfusion injury in animals.(2)In the MCAO model is established after 2 hours of ischemia and reperfusion for 24 hours,were observed neurological deficits,infarct proportion and morphological features which were performed in sham-operated group,I/R group,low dose group,middle dose group and high dose group.The effects of the calycosin in the rat cerebral ischemia reperfusion injury were evaluated by the comparison of animal level experiments.(3)The relationship between the expression of autophagy related protein p62 and NBR1 in the rat brain tissue and the level of apoptotic factor Bcl-2 and inflammatory factor TNF-α was further detected by western blotting,quantitative real-time PCR and immunohistochemistry.Results: On the 14 day after pretreatment,calycosin significantly reduced neuronal damage in cerebral ischemia-reperfusion,significantly improved neurological function score and reduced infarct volume and brain edema in rats with focal cerebral ischemia-reperfusion.We observed that after cerebral ischemia-reperfusion,the expression of p62,NBR1 and Bcl-2 protein increased significantly,and TNF-α level decreased significantly through western blotting,RT-PCR and immunohistochemistry.However,in the MCAO,the expression of p62,NBR1 and Bcl-2 upregulated significantly with the increase of the concentration of the calycosin,and the level of TNF-α was also downregulated,which was also correlated with the dosage of calycosin.Conclusion: Our study suggests that phytoestrogens calycosin play a protective effect by p62,NBR1 and Bcl-2 upregulated and TNF-α downregulated of rat brain tissues on ischemia reperfusion injury of rat neurons and the mechanisms maybe associated with its antiautophagic,anti-apoptotic and anti-inflammatory action.These results provide a new direction for the treatment of stroke.