| Background:Alzheimer’s Disease(AD)is a serious disease of the central nervous system,mainly manifested by cognitive impairment such as learning and memory impairment.With the aging of the population,the incidence of AD bringing a heavy burden to the society and family,and the current drug treatment can only alleviate the pathological process of patients.Traditional Chinese medicine has the advantages of multi-component,multi-target and multi-channel in the treatment of diseases,and has achieved good results in the treatment of clinical AD.Asafoetida(ASF)is an oleoresin obtained from the rhizomes of Ferula assafoetida L.plants.It is Xin Er Neng San and has the effect of promoting blood circulation and removing blood stasis.Modern research has proved that it has antioxidant and neuroprotective properties effect,which is closely related to neurological diseases.However,the efficacy and further mechanisms of ASF in AD treatment remain unclear.Further experimental research may provide clues and basis for the development of new drugs for AD treatment.Therefore,this study intends to further explore the efficacy and mechanism of ASF in the prevention and treatment of AD,and provide a basis for the clinical application of ASF and the development of new drugs.Methods:1.Study on the neuroprotective effect of ASF extracts on scopolamine-induced cognitive impairment model mice with reduced learning and memory ability,pathological damage to hippocampus and neuronal degeneration.(1)Animal group administration and model establishment:Male SPF C57BL/6 mice were randomly divided into 5 groups with 15 mice in each group,control group,scopolamine model group,low(37.5 mg/mL),medium(75 mg/mL)and high-dose(150 mg/mL)groups.The administration group was given different concentrations of ASF extracts,and the control group and the model group were given equal volume of normal saline for 14 consecutive days.Mice in each group were intraperitoneally injected with scopolamine(3 mg/kg)15-20 min before behavioral study to construct AD models of cognitive impairment.(2)Behavioral evaluation:Morris water maze test,platform jumping test,and novel obj ect recognition test were used to evaluate the improvement effect of ASF extracts on the learning and memory ability of scopolamine-induced AD model mice;(3)Evaluation of pathological indicators:Nissl staining was used to observe the morphological changes and neuronal damage of hippocampal neurons in AD mice;biochemical methods were used to detect Ach content,SOD activity,GSH-PX activity and MDA level in the hippocampus of mice;RT-qPCR was used to detect the expression level of AchE mRNA;TUNEL staining was used to detect the apoptosis of neurons in the hippocampus of mice;Western blot was used to detect the protein expression levels of Nrf2,Bcl-2 and Bax.2.Taking oxidative damage and neuronal apoptosis as the breakthrough point,to explore the protective effect of ASF extracts on oxidative damage and neuronal apoptosis of PC 12 cells stimulated by H2O2.(1)In vitro culture of PC 12 cells:H2O2 intervenes PC 12 cells to build an in vitro model of oxidative damage.(2)The CCK-8 method was used to detect the effect of ASF extracts on the activity of PC12 cells.(3)Detection of reactive oxygen species(ROS)levels in PC12 cells damaged by H2O2 by DCFH-DA probe method.(4)Biochemical method was used to detect the level of representative indicators of oxidative stress in PC 12 cells damaged by H2O2.(5)The apoptosis rate of PC 12 cells damaged by H2O2 was detected by flow cytometry,and the protein expression levels of apoptotic cells were detected by Western blot and RTqPCR.3.The chemical constituents of ASF were identified by liquid chromatography-mass spectrometry(UHPLC-Q-Orbitrap MS).4.The network pharmacology method was used to explore the targets of ASF in improving H2O2-induced oxidative damage and neuronal apoptosis in PC 12 cells.5.Western blot,RT-qPCR and immunofluorescence were used to verify the expression levels of PI3K,AKT,GSK3β,Nrf2 and downstream signaling pathway-related proteins in PC 12 cells intervened by H2O2,and to clarify the potential mechanism of ASF in the treatment of AD.Results:1.In vivo results(1)Behavioral test results:The results of the Morris water maze test showed that after administration of low,medium and high doses of ASF extracts,the number of platform crossings and the time spent on the target platform of AD model mice were significantly prolonged(P<0.05),the dwell time in the relative target quadrant was significantly shorter(P<0.05).The results of platform jumping experiment and novel object recognition experiment showed that each dose group of ASF extracts reduced the number of mistakes,escape latency and recognition index of mice in the model group(P<0.05).(2)The results of biochemical and RT-qPCR assays showed that compared with the model group,ASF extracts could significantly increase the SOD activity,GSH-PX activity and Ach content in the hippocampus of cognitively impaired mice(P<0.05),decreased AchE activity and MDA level(P<0.05);at the same time,it could reduce AchE mRNA expression level(P<0.05).(3)The results of Nissl staining showed that the number of Nissl bodies in the CA1 and CA3 regions of the mice in the model group was significantly reduced,the arrangement of neurons in the hippocampus was disordered,and the nucleoli of neurons were pyknotic and hyperchromatic.The low,medium and high dose groups of ASF extracts improved the pathological damage of the hippocampus of mice to different degrees.(4)TUNEL results showed that compared with the control group,the scopolamine model group showed positive staining,indicating that it can promote neuronal apoptosis.Compared with the model group,the positive expression of TUNEL in the brain tissue of the cognitively impaired mice in the low,medium and high extract groups of ASF extracts was significantly attenuated.(5)WB results showed that compared with the mice in the control group,the expression level of Bcl-2 protein in the hippocampus of the mice in the model group was down-regulated(P<0.05),and the protein expression of Bax was up-regulated.The protein expression level of-2 was up-regulated(P<0.05),and the protein expression of Bax was down-regulated(P<0.05).2.In vitro results(1)The results of CCK-8 activity test showed that the optimal administration concentration of ASF extracts to treat PC 12 cells was 0.1,1,10 μg/mL,and the optimal damage concentration of H2O2 to intervene PC 12 cells was 175 μM.(2)The detection results of DCFH-DA probe method showed that compared with the control group,the ROS fluorescence intensity of the mice in the model group was significantly increased.There were different degrees of decline(P<0.05).(3)The results of biochemical assay showed that compared with the control group,the activities of SOD(P<0.05)and GSH-PX(P<0.05)in the model group were significantly decreased,and the level of MDA was significantly increased(P<0.05);Compared with the other groups,the activities of SOD(P<0.05)and GSH-PX(P<0.05)were significantly increased,and the level of MDA was significantly decreased(P<0.05)in each dose group of ASF.(4)In the flow cytometry detection,the apoptosis rate of the model group was significantly higher than that of the control group(P<0.05).3.UHPLC-Q-Orbitrap MS was used to identify 31 main components in the ASF.4.The results of network pharmacology showed that PI3K,Akt,GSK3β,Nrf2,HO-1 may be the main effector targets of ASF extracts to exert neuroprotective function.3.Network pharmacology research and validation results.5.WB and RT-qPCR results showed that the expression of PI3K/AKT/GSK3β/Nrf2/HO-1 pathway-related proteins in H2O2-injured PC 12 cells changed significantly compared with the blank group.The expression levels of PI3K,phosphorylated AKT,Nrf2,and HO-1 in the middle and high dose groups were increased(P<0.005),and the expression level of phosphorylated GSK3β was significantly decreased(P<0.05).The results of immunofluorescence showed that Nrf2 was mainly located in the cytoplasm of normal PC 12 cells before modeling with H2O2.Compared with the model group,the low,medium and high dose groups of ASF extracts could promote PC 12 cells to different degrees nuclear translocation.Conclusions:In vivo studies,ASF extracts can significantly improve learning and memory ability,neuronal degeneration and pathological damage,slow down oxidative stress and neuronal apoptosis,and improve brain tissue damage in scopolamine-induced cognitive impairment mice;in vitro studies,ASF can play a neuroprotective role by regulating the PI3K/AKT/GSK3β/Nrf2/HO-1 pathway,slowing down oxidative stress and neuronal apoptosis. |
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