| Objective1.Prediction of effective compounds in Salvia miltiorrhiza for the treatment of Alzheimer’ s disease(AD)use a network pharmacology approach.2.To examine the efficacy of tanshinone ⅡA(Tan ⅡA)in APP/PS1 doubletransgenic mice and to explore the possible mechanisms of its action.3.Combining the pathway results predicted by the group’ s previous studies and enrichment analysis,using molecular docking techniques to validate targets that both play a key role in the drug-target-disease network and bind well to Tan ⅡA.4.To explore the effect of Tan ⅡA on the screened A β-degrading enzymes and use it as an entry point to explore the potential mechanism of action of Tan ⅡA in the treatment of AD from the perspective of endoplasmic reticulum stress(ER stress).5.In vitro experiments were conducted to further validate the effects of Tan ⅡA on ER stress and A β degradation and to explore the mechanisms involved in its effects on AD.Methods1.In this study,we screened the effective compounds in Salvia miltiorrhiza through TCMSP database,found the targets corresponding to these effective compounds through TCMSP database and Uniprot database,then collected the AD-related targets through GeneCard database and OMIM database,found the intersection targets and their corresponding effective compounds in Excel.The PPI network of intersecting targets and active compounds was constructed by Cytoscape software,and the results were visualized.The active compound with the strongest association with AD was selected as the research object according to the degree value in Cytoscape,and the intersection of Tan ⅡA with the corresponding target of AD was found by TCMSP database and PharmMapper database.The results were visualized by Cytoscape software,GO and KEGG were performed on the intersecting targets by DAVID database,and the results were visualized by the GENE DENOVO website.2.Ten wild-type(WT)mice were used as normal control group and 30 transgenic(APP/PS1)mice were randomly divided into three experimental groups(n=10):model group,Tan ⅡA low-dose group(10 mg/kg)and Tan ⅡA high-dose group(20 mg/kg).The mice were reared until 6 months of age and gavaged at a volume of 0.1 ml/10 g for 8 weeks,followed by behavioral and 18F-FDG Micro-PET/CT assays to initially determine the efficacy of Tan ⅡA on APP/PS1 double transgenic mice.At the end of the assay,7 mice in each group were randomly selected to be anesthetized with 10%chloral hydrate and then their brains were severed.In addition,3 mice in each group were anesthetized and perfused with 4%paraformaldehyde,and the brain tissues were removed and prepared into paraffin sections after fixation,and the kit assay,Western Blot assay and Nissl staining were used to verify the effect of Tan ⅡA on neuronal oxidative stress,neuronal apoptosis,cholinergic system and neurodegeneration in APP/PS1 mice.3.Western Blot was used to detect the protein expression of ER stress and key targets in the UPR signaling pathway,while immunofluorescence was used to detect the expression of Bip,a molecular chaperone of ER stress,observing the effect of Tan ⅡA on ER stress in the brain of APP/PS1 mice.4.ThT staining and immunohistochemistry were used to detect the expression of Aβ to observe the effect of Tan ⅡA on Aβ in the brain of APP/PS1 mice.The molecular structure of Tan ⅡA monomer as docking ligand was obtained from the TCMSP database,and then the target protein structure of A β-degrading enzymes as docking acceptor was obtained from the RSCB PDB database.The semi-flexible docking of A β-degrading enzymes with Tan ⅡA was performed using the CB-Duck online analysis website,and the conformational parameters generated by the docking were recorded and the visualization results were downloaded.The expression of the key Aβdegrading enzymes IDE and NEP in the cerebral cortex and hippocampus was examined by Western Blot and immunofluorescence to investigate the efficacy of Tan ⅡA in the treatment of AD.5.Cellular experiments were performed using 20 μ M of A β 1-42 oligomer to HT22 cells,and Aβ1-42-induced HT22 cells were intervened with 0.1,1 and 10 μM concentrations of sodium tanshinone ⅡA sulfonate(STS).The possible pharmacodynamic mechanism of Tan ⅡA action on AD was further verified by TUNEL staining,flow cytometry,Western Blot and kit assay.Results1.The network pharmacology method was used to screen out 36 active compounds from Salvia miltiorrhiza,which corresponded to 156 targets,9605 key targets of AD and 72 intersecting targets with the active compounds.The PPI network generated by these 39 intersecting targets contains 38 nodes and 264 edges,and the enrichment analysis shows that Tan ⅡA is involved in the regulatory mechanism of AD and various neurodegenerative diseases,and this is probably related to the fact that Tan ⅡA can act on the biological process of Aβ.2.In the Morris water maze test,compared with the WT group,the APP/PS1 group had a significantly longer escape latency(P<0.001),a significantly lower number of platform crossings(P≤0.001),and a significantly shorter dwell time in the target quadrant(P<0.001),and compared with the APP/PS1 group,both the low and high dose groups of TanⅡA had a significantly shorter escape latency(P<0.001),and although the number of platform crossings showed an increasing trend,the differences were not statistically significant(P>0.05,P>0.05),the dwell time in the target quadrant was prolonged(P<0.05,P<0.05).In the open-field test,the number of times the APP/PS1 group entered the central area was significantly reduced compared to the WT group(P<0.001),and the distance to the central area activity was also significantly shortened(P<0.001),and the number of times the Tan ⅡA low and high dose groups entered the central area was increased compared to the APP/PS1 group(P<0.05,P<0.01),there was a tendency to increase the distance traveled for central area activity,but the difference was not statistically significant(P>0.05,P>0.05).In the novelty object recognition test,the novelty object recognition rate was reduced in the APP/PS1 group compared to the WT group(P<0.05),and the novelty object recognition rate was increased in the Tan ⅡA low-dose group compared to the APP/PS1 group(P<0.05),but the difference was not statistically significant in the high dose(P>0.05).The results of 18F-FDG Micro-PET/CT visual analysis showed that the model group exhibited a bilateral largely symmetrical decrease in the level of glucose metabolism in the brain compared with the WT group,and a bilateral largely symmetrical increase in the level of glucose metabolism in the brain of the Tan ⅡA high-dose group compared with the APP/PS1 group,but the difference was not significant in the low-dose group.3.In the kit assay of key factors of oxidative stress,compared to the WT group,the APP/PS1 group showed increased MDA content(P<0.001)and decreased SOD and GSH-Px activities(P<0.001,P<0.001),and compared to the APP/PS1 group,Tan ⅡA low and high doses showed decreased MDA content(P<0.001,P<0.001)and SOD and GSH-Px activities increased(P<0.01,P≤0.001;P<0.001,P<0.001).In the apoptosis key protein WB assay,compared with the WT group,Bax/Bcl-2 and Caspase-3 levels were significantly increased in the APP/PS1 group(P<0.001,P<0.05),p-AKT levels were decreased(P<0.05),PI3K levels were decreased but the difference was not statistically significant(P>0.05),compared with the APP/PS1 group,Bax/Bcl-2(P<0.001,P<0.001)and Caspase-3 levels(P<0.05,P(0.05)were significantly lower in the Tan ⅡA low and high dose groups,and PI3K levels were increased but not statistically different in the low dose group(P>0.05,P<0.05),and p-AKT levels increased in the low-dose group but not in the high-dose group(P<0.05,P>0.05).4.In the kit assay for cholinergic-related factors,AChE levels increased in the APP/PS1 group compared to the WT group,while Ach and ChAT levels decreased(P<0.05,P<0.05,P<0.05),and AChE levels decreased in the Tan ⅡA low and high dose groups compared to the APP/PS1 group,although there was no statistical difference(P>0.05,P>0.05),Ach levels increased in the high dose group but not in the low dose group(P>0.05,P<0.05),and ChAT levels showed a significant trend of increase but the difference was not statistically significant(P>0.05,P>0.05).In the WB assay of neurotrophic factors,BDNF and PSD95 levels were significantly lower in the APP/PS1 group compared to the WT group(P<0.001,P<0.01),and BDNF levels were increased in the Tan ⅡA low and high dose groups compared to the model group(P<0.01,P<0.01),and PSD95 levels were increased(P<0.01,P<0.01).In Nissl staining,the number of Nissl bodies in the cortex and hippocampus of the APP/PS1 group was significantly reduced,mostly in the form of vacuoles and lighter in color compared with the WT group,and the Nissl bodies in the cortex and hippocampus of the Tan ⅡA low and high dose groups were clearly discernible,tiger-like,darkly stained and significantly increased in number compared with the APP/PS1 group.5.In the WB assay of key proteins for ER stress and UPR,compared with the WT group,Bip,p-PERK,ATF6,p-IRE1α and CHOP levels were increased in the APP/PS1 group(P<0.001,P≤0.001,P<0.001,P<0.001,P<0.001,P<0.001),and compared with the APP/PS1 group,the Tan ⅡA low and high dose groups had lower Bip levels(P≤0.001,P≤0.001),and lower p-PERK levels(P≤0.001,P<0.01),decreased ATF6 levels(P<0.01,P≤0.001),decreased p-IRE1α levels(P<0.001,P<0.001),decreased CHOP levels(P<0.001,P<0.001),In the immunofluorescence assay,the fluorescence intensity of Bip was higher in the APP/PS1 group compared to the WT group,and became lower in the Tan ⅡA low and high dose groups compared to the APP/PS1 group.6.In ThT staining,the green fluorescent signal was stronger in the APP/PS1 group compared with the WT group,and the fluorescent signal was reduced in both the Tan ⅡA low and high dose groups compared with the APP/PS1 group.In the A β immunohistochemical assay,the tan deposition was more in the APP/PS1 group compared with the WT group,and the tan deposition was reduced in both the Tan ⅡA low and high dose groups compared with the APP/PS1 group.The molecular docking results showed that the absolute values of the five conformation scores of the semi-flexible docking of the macromolecular receptor proteins IDE and NEP with the ligand Tan ⅡA were≥ 5,indicating that both IDE and NEP could bind well to Tan ⅡA.In the WB assay,compared with the WT group,the IDE and NEP levels were decreased in the APP/PS1 group(P<0.05,P<0.01),and compared with the APP/PS1 group,the IDE and NEP levels were increased both two dose groups of TanⅡA(P<0.05,P<0.05;P≤0.001,P<0.05).In the immunofluorescence assay,the IDE and NEP fluorescence intensities were reduced in both cortex and hippocampus in the APP/PS1 group compared with the WT group,and the IDE and NEP fluorescence intensities were increased in both cortex and hippocampus in the Tan ⅡA low and high dose groups compared with the APP/PS1 group.7.In cell experiments we pretreated 20 μM Aβ1-42-induced HT22 cells with 0.01,0.1,1 and 10 μM concentrations of STS,and cell survival was significantly reduced in the Aβ group compared to the control group(P<0.001),and there was no statistical difference in the STS0.01 μM group compared to the A β group(P>0.05).Cell survival increased in the STS0.1,1 and 10 μM groups(P<0.001,P<0.001,P<0.001).Microscopic observation of cell growth and morphological characteristics of each group showed that compared with the control group,the Aβ group had sparse cell density,more suspended cells,wrinkled and rounded cells,solidified nuclei,condensed chromatin and irregular morphology,and compared with the Aβ group,the STS0.1,1 and 10 μM groups had increased cell density,extended cells against the wall and regular morphology.8.In TUNEL staining,the positive fluorescence was strong and intensive in the A β group compared to the control group,and the number of positive fluorescence was reduced in the STS0.1,1 and 10 μM groups compared to the Aβ group.In flow cytometry detection of apoptosis,the rate of apoptosis was increased in the Aβ group compared with the control group(P<0.01),and both early and late apoptotic cells were significantly increased(42.4%;23.8%)and normal cells were decreased(33.5%),compared with the Aβ group,the rate of apoptosis was decreased in the STS0.1,1 and 10 μM groups(P<0.05,P≤0.001,P<0.05),and there was a decrease in both early and late apoptotic cells(34.8%,25.3%,13.4%;19.0%,8.4%,12.2%)and an increase in normal cells(46.1%,66.3%,74.0%)in each administration group.In the WB assay of apoptosis-related proteins,Bax/Bcl-2 and Caspase-3 levels were significantly increased(P<0.001,P<0.001)and p-AKT levels were decreased(P<0.01)in the Aβ group compared to the control group,and there was no statistical difference between groups despite a trend in PI3K levels;compared to the Aβ group Bax/Bcl-2(P<0.001,P<0.001,P<0.001)and Caspase-3 levels(P<0.001,P<0,001,P<0.001)were significantly lower in the STS0.1,1 and 10 uM groups compared to the Aβgroup,and p-AKT levels were increased in all 3 dosing groups(P<0.01,P<0.05,P<0.05).In the flow cytometry detection of ROS,intracellular ROS levels were increased in the Aβ group compared to the control group(P<0.001),with a stronger intracellular DCF signal(63.7%)and a significant shift of the main peak of ROS to the right side of the baseline,and a decrease in intracellular ROS levels in the STS0.1,1 and 10 1M groups compared to the Aβ group(P<0.001,P<0.001,P<0.001),the intracellular DCF signal was attenuated in each administration group(33.3%,32.4%,29.6%),and the main peak of ROS shifted to the left side of the baseline.In the oxidative stress-related factor kit assay,MDA levels were significantly increased in the Aβ group compared to the control group(P<0.001),while SOD and GSH-Px levels were significantly decreased(P<0.001,P<0.01),and compared to the Aβ group,MDA levels in the STS0.1,1 and 10 μM groups were decreased(P<0.001,P<0.001,P<0.001),SOD levels increased in the STS1 and 10 μM groups(P<0.05,P<0.05),while SOD levels in the STS0.1 μM group showed an increasing trend but were not statistically significant(P>0.05),and GSH-Px levels in the STS0.1,1 and 10 μM groups were not statistically significant although they all slightly increased(P>0.05,P>0.05,P>0.05).9.In the WB assay of ER stress and UPR-related proteins,compared with the control group,there were increased levels of Bip,p-PERK,p-eIF2α,p-IREla and XBP1s in the Aβ group(P<0.01,P<0.001,P<0.001,P<0.001,P<0.001,P<0.001),although there was a significant trend of higher CHOP levels,there was no statistical difference yet(P>0.05),ATF6 levels were not statistically different(P>0.05),and Bip levels were reduced in the STS1 and 10 μM groups compared to the Aβ.group(P<0.05,P-0.001),no statistical difference in the 0.1 μM group(P>0.05),and no statistical difference in the STS1 and 10 μM group had reduced p-PERK levels(P<0.01,P<0.05),the 0.1 μM group was not statistically different(P>0.05),and the STS0.1,1 and 10 μM groups had significantly reduced p-eIF2 a levels(P<0.001,P<0.001,P<0.001),ATF6 levels were not statistically different in the STS0.1,1 and 10 μM groups(P>0.05,P>0.05,P>0.05),and p-IRE1α levels were reduced in the STS0.1,1 and 10 μM groups(P<0.01,P<0.001,P<0.001),and XBPls levels were reduced in the STS0.1,1 and 10 μM groups(P<0.001,P<0.001,P<0.001),and although there was a significant trend toward lower CHOP levels in the STS0.1,1 and 10 μM groups,the differences were not yet statistically significant(P>0.05,P>0.05,P>0.05).10.In WB assay,compared with the control group,A β1-42 levels were significantly higher in the Aβ group(P<0.001),especially at 38 to 42 kDa,and IDE and NEP levels were decreased(P<0.01,P<0.001).Compared with the A β group,the A β 1-42 levels were significantly lower in the STS0.1,1 and 10 μM groups(P<0.001,P<0.001,P<0.001),and IDE levels were statistically different in STS0.1 and 1 μM groups(P<0.01,P<0.05),and NEP levels were significantly higher in all dosing groups(P<0.001,P<0.001,P<0.001).Conclusion1.The predicted results of the network pharmacology study suggest that Tan ⅡA is the most effective compound in Salvia miltiorrhiza with the closest connection to AD,and it acts on AD through a multi-target and multi-pathway pharmacodynamic pathway,which is likely to be related to the mechanism that Tan ⅡA acts on the biological process of Aβ.2.Animal experiments have verified that Tan ⅡA can improve the learning memory ability,cholinergic system and neurodegeneration in APP/PS1 double transgenic mice,and the mechanism may be related to the upregulation of Ach,BDNF and PSD95.3.Both animal and cellular experiments verified that Tan ⅡA could reduce oxidative stress and neuronal apoptosis in AD model,and the mechanism may be related to the upregulation of SOD,GSH-Px,PI3K,p-AKT and Bcl-2,and downregulation of ROS,MDA,Bax and Caspase-3.4.Both animal and cellular experiments verified that Tan ⅡA can alleviate ER stress in AD model,and the mechanism may be related to downregulation of Bip,p-PERK,p-eIF2α,ATF6,p-IRE1 α,XBP1s and CHOP.5.The molecular docking results predicted that the receptor proteins IDE and NEP could bind well to the ligand Tan ⅡA,and both animal and cellular experiments verified that Tan ⅡA could reduce the expression level of A β in AD model,and the mechanism might be related to the upregulation of IDE and NEP. |
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