The Mechanism Of Transient Global Cerebral Ischemic Tolerance Mediated By Rhodiola Sacra Through Activating AMPK/Nrf2 Signaling Pathway In Rats

Background and PurposeAcute cerebral ischemic events are common in clinic and seriously endanger the health of human.Intravenous thrombolysis and/or intravascular therapy do benefit some patients,but their clinical application is limited to some extent due to the narrow time window of treatment and the risk of bleeding.Although the existing neuroprotective drugs for acute cerebral ischemia are effective in experimental studies,the results in clinical trials are not satisfactory.Therefore,more attention has been paid to the prevention and treatment of acute cerebral ischemia with traditional Chinese medicine.Rhodiola sacra(RS)is a perennial herb of rhodiola sacra species,crassulaceae,grown at alpine grassland and rock joints between2700-4600 m altitude in Tibet and northwest area of Yunnan province in China.Our previous study has confirmed that adult male Wistar rats was continuously given RS for 28 d before tGCI(RS0),given RS for 7 d after tGCI(RS1),or given RS for 28 d before tGCI combining with 7 d after tGCI(RS3),can significantly reduce the death and degeneration of neurons in the hippocampus CA1 area after ischemia.However,the specific mechanism of RS exerting neuroprotective effect is still unclear.There are some evidences indicate that oxidative stress plays an important role in cerebral ischemia-reperfusion.Nuclear factor erythroid-2-related factor 2(Nrf2)is one of the key regulators of endogenous redox system,which controls the expression of series of downstream antioxidant genes.Some studies suggest that Nrf2 may participate in the antioxidant of RS on cerebral ischemia.Nrf2 is regulated by a variety of factors,among which adenosine monophosphate activated protein kinase(AMPK)is closely related to the activity of Nrf2.Based on our previous research,this project aims to investigate the neuroprotective effect of RS on tGCI in adult rats.We also attempt to explore the potential mechanism by which RS prevents tGCI-induced neuronal death via activating AMPK,promoting nuclear translocation of Nrf2 and thereby alleviating oxidative stress in the hippocampus CA1 after tGCI.Material and methodsIn this study,adult male Wistar rats with weight of 220-280 g and age of 7-8 w were selected.The tGCI model was conducted according to the modified Pulsinelli classical fourvessel occlusion method.RS or Normal Saline(NS)with the same volume as RS was continuously administered by gavage for 28 d before tGCI,7 d after tGCI,or 28 d before tGCI combining with 7d after tGCI.The number and morphological changes of survival cells,neurons and glial cells in CA1 area were detected by Nissl,Fluoro-Jade B staining and immunohistochemistry.By using Morris water maze,we evaluate the spatial learning and memory ability of rats.MDA content in CA1 region of each group was detected by MDA kit.The location and distribution of Nrf2,AMPK and p-AMPK of CA1 were detected by immunohistochemistry and immunofluorescent study.Through Western Blotting,we detect the expression of Nrf2,p-AMPK and AMPK in CA1 region of each group.After silencing Nrf2 by bilateral hippocampal injection of si Nrf2,the expression of Nrf2 and HO-1 in the above groups were detected by Western Blotting.The binding sites of salidroside(the active components of RS)and AMPK were predicted by molecular docking software.Co-IP was used to detect the relationship between AMPK and Nrf2 in the CA1 region of rats.The activation of AMPK was inhibited by Compound C through intracerebroventricular injection.With co-immunoprecipitation,we further examined the interaction between AMPK and Nrf2 in rats after tGCI with or without rhodiola sacra treatment.Result1.RS preconditioning(RS0),with a dose of 2.4 g/kg/d,28 consecutive days before tGCI,RS postconditioning(RS1),with a dose of 2.4 g/kg/d,7 consecutive days right after tGCI,and RS pre-conditioning combining with postconditioning(RS3)in adult male Wistar rats exert neuroprotective effects through promoting neuronal survival and alleviating glial cell activation in CA1 after tGCI.2.Morris water maze results showed RS significantly improves the spatial learning and memory ability of rats after tGCI.3.RS treatment increases the expression of Nrf2 in the nucleus of hippocampal CA1 region after tGCI.MDA content in hippocampal CA1 area was significantly increased after tGCI in rats,and significantly decreased after RS treatment.After injection si Nrf2,the expression of Nrf2 and HO-1 was significantly reduced.4.Molecular docking test results showed that there was an interaction between salidroside and AMPK,the active component of Rhodiola rosea,while Co IP results showed an interaction between P-AMPK and Nrf2.RS treatment significantly increases the expression of p-AMPK in CA1 after tGCI.After injection with AMPK inhibitior,Compound C,the effects of RS on the p-AMPK and Nrf2 expression were counteracted.Conclusion1.RS alleviates neuronal death and glial cell activation in the hippocampal CA1 after tGCI in adult rats.2.RS improves cognitive function of rats after tGCI.3.RS alleviates tGCI-induced neuronal injury through increasing the expression and promoting nuclear translocation of Nrf2 in CA1 area after tGCI.4.RS plays a neuroprotective role by activating the AMPK/Nrf2 pathway and promoting nuclear translocation of Nrf2 in the hippocampal CA1 area after tGCI.