The Effect Of Imperatorin On Mitochondrial Smac/DIABLO Signaling Pathway In Mouse Model Of Alzheimer’s Disease Induced By Aβ_1-42

Objective:To study the effect of imperatorin(IMP)on second mitochondrial-derived caspase activator(Smac/DIABLO)signaling pathway-related proteins in Aβ1-42 induced Alzheimer’s disease(AD)model mice,and to explore the neuroprotective effect of IMP on Aβ1-42 induced AD model mice.Methods:1.To observe the effect of Aβ1-42 on Smac/DIABLO signaling pathway-related proteins:the AD model of isolated brain slices was established by incubating with 1μM,10 μM and 20 μM Aβ1-42 for 12 h,and the relevant indicators were detected after 12 h incubation of brain slices.After the brain slices were homogenized,the damage degree of the brain slices was observed by measuring the activity of lactate dehydrogenase(LDH).The protein content of Smac/DIABLO and X-linked inhibitor of apoptosis protein(XIAP)in the brain slices was detected by ELISA,and the activities of caspase-3 and caspase-9 were detected by the activity detection kit of caspase-3 and caspase-9.The AD mouse model was established by intraventricular injection of Aβ1-42,and the protein expression of Smac/DIABLO,XIAP,caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,poly adenosine diphosphate ribose polymerase(PARP)and cleaved PARP in the cytoplasm of mouse cerebral cortex were detected by Western Blot.2.To study the effect of IMP on the expression of Smac/DIABLO signaling pathway-related proteins in Aβ1-42 induced AD mouse model.AD mouse model was established by intraventricular injection of Aβ1-42.Imperatorin(2.5 and 5.0 mg·kg-1)was intraperitoneally injected on the day of modeling,once a day for 13 consecutive days.The sham operation group and AD model group was intraperitoneally injected with vehicle 10 mL·kg-1 at the same time point.On the 14th day after administration,mouse cerebral cortex tissue was isolated.The protein expression of Smac/DIABLO,XIAP,caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,PARP and cleaved PARP in the cytoplasm of mouse cerebral cortex were detected by Western Blot;the protein content of Smac/DIABLO and XIAP in mouse cerebral cortex were detected by ELISA;the protein activities of caspase-3 and caspase-9 in mouse cerebral cortex were detected by caspase-3 and caspase-9 activity detection.Results:1.The results of LDH detection in brain slices showed that compared with the normal control group,the LDH activity of Aβ1-42 group at1μM,10 μM and 20 μM were all significant increase(P<0.05,P<0.01).The protein content of Smac/DIABLO and XIAP in brain slices was detected by ELISA.The results showed that compared with the normal control group,the protein content of Smac/DIABLO in the 10 μM and 20 μM Aβ1-42 groups was significantly increased(P<0.01),while the protein content of XIAP were significantly decreased(P<0.01).The activity results of caspase-3 and caspase-9 in brain slices showed that compared with the normal control group,the activities of caspase-3 and caspase-9 in the 1 μM,10 μM,20 μM Aβ1-42 group were significantly increased(P<0.01).The protein expression of Smac/DIABLO,XIAP,caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,PARP and cleaved PARP in the cytoplasm of AD model mouse prepared by Aβ1-42 were detected by Western Blot.The results showed that compared with the sham operation group,the protein expression of Smac/DIABLO,cleaved caspase-3,cleaved caspase-9 and cleaved PARP and the ratio of cleaved caspase-3/caspase-3,cleaved caspase-9/caspase-9 and cleaved PARP/PARP in AD model group were significantly increased(P<0.05,P<0.01),while the protein expression of XIAP was significantly decreased(P<0.05).2.The effect of imperatorin on the expression of Smac/DIABLO,XIAP,caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,PARP and cleaved PARP in the cytoplasm of Aβ1-42 induced AD model mouse.The results showed that compared with the AD model group,the protein expression of Smac/DIABLOand the ratio of cleaved caspase-3/caspase-3 in the imperatorin(2.5 and 5.0 mg·kg-1)treatment group were significantly decreased(P<0.05,P<0.01);the protein expression of cleaved caspase-3,cleaved caspase-9,cleaved PARP and the ratios of cleaved caspase-9/caspase-9 and cleaved PARP/PARP in the 5.0 mg·kg-1 imperatorin drug group were significantly decreased(P<0.05,P<0.001),while the protein expression of XIAP in the 5.0 mg·kg-1 imperatorin group was significantly increased(P<0.01).The protein content of Smac/DIABLO and XIAP in mouse cerebral cortex was detected by ELISA.The results showed that compared with the normal control group,the protein content of Smac/DIABLO in AD model group was significantly increased(P<0.05),while XIAP protein content was significantly decreased(P<0.05);compared with AD model group,Smac/DIABLO protein content in imperatorin(2.5 and 5.0 mg·kg-1)treatment group was significantly decreased(P<0.05,P<0.01),while XIAP protein content was significantly increased(P<0.05).The activity of caspase-3 and caspase-9 in mouse cerebral cortex was detected by the activity detection of caspase-3 and caspase-9.The results showed that compared with the sham operation,the activities of caspase-3 and caspase-9 in the AD model group were significantly increased(P<0.01);compared with the AD model group,the protein activities of caspase-3 and caspase-9 in the imperatorin(2.5 and 5.0 mg·kg-1)treatment group were significantly decreased(P<0.01).Conclusion:Imperatorin can down-regulate the protein expression of Smac/DIABLO in the cytoplasm of cerebral cortex in Aβ1-42 induced AD model mouse,up-regulate the protein expression of XIAP,inhibit the activation of caspase-3 and caspase-9,and alleviate the nerve damage of AD.This may be one of the mechanisms by which imperatorin exerts neuroprotective effects in AD.