| Alzheimer's disease?AD?is a chronic neurodegenerative disorder with neither definitive pathogenesis nor effective therapy so far.AD pathogenesis is characterized by amyloid-?plaques deposition,tau hyperphosphorylation,neuroinflammation,mitochondrial dysfunction and oxidative stress.Amyloid-?protein?A??is formed by different peptide segments,in which A?42 has higher neurotoxicity than A?40 because of its strong ability to induce cell apoptosis,inflammatory reactions and oxidative stress.In Western medicine,licensed drugs are used to improve cognitive functions and single-target pharmacotherapy exhibit limited efficacy clinically,up to now,there has no effective treatment for AD.The effective ingredients of traditional Chinese medicine must be transported across the blood-brain barrier to the target parts of CNS disords.Blood-brain barrier?BBB?is a selective border which exists between brain tissue and blood and consists of astrocyte podocytes,brain microvascular endothelial cells and their basement membranes.BBB contains many kinds of specific transporters,such as P-glycoprotein?P-gp?,breast cancer resistance protein?BCRP?,etc.As efflux pumps,these transporters can discharge substrates from the brain and protect the brain from toxic substances.On the other hand,by restricting the transport of certain drugs to the brain,reducing the therapeutic effect of drugs,BBB plays an important role in maintaining the balance of the brain.Danshen is the dried root and rhizome of Salvia miltiorrhiza Bunge and used extensively in Alzheimer's disease.However,the therapeutic mechanism and the transportation across BBB of Danshen are still unclear.Therefore,this paper conducted three studies:1.UHPLC-QTOF/MS-based metabolomics investigation for the protective mechanism of Danshen in Alzheimer's disease cell modelObjectives:To investigate potential biomarkers for AD and elucidate the protective mechanism of Danshen in AD cell model.Methods:A?1-42 oligomers were prepared and used to induce human brain microvascular endothelial cell injury to establish AD cell model,and to evaluate the protective effect of Danshen on AD cell model.The potential metabolic biomarkers of AD cell model were screened out by ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry combined with partial least squares discriminant analysis?PLS-DA?.The related metabolic pathways were analyzed and integrated,and the protective mechanism of Danshen on AD cell model was analyzed.Results:?1?A?1-42 oligomer was successfully prepared by incubating the A?1-42polypeptide monomer at 4?for 24 hours.The AD cell model was established by inducing hBMEC injury with 10?M A?1-42 oligomer.100 ug/mL Danshen extract could significantly improve the cell viability of AD cell model.?2?Thirty-three distinct potential biomarkers were screened out and considered as potential biomarkers corresponding to AD,which were mostly restored back to normalcy in Danshen pre-protection group.?3?It was found that AD was closely related to disturbed alanine aspartate and glutamate metabolism,glutathione metabolism,arginine and proline metabolism,tryptophan metabolism,pantothenate and CoAbiosynthesis,histidinemetabolism,phenylalaninemetabolismand glycerophospholipid metabolism in AD cell model.Conclusion:The AD cell model induced by A?1-42 oligomer was closely related to the disorders of the above metabolic pathways.The protective mechanism of Danshen on cell model may be related to the regulation of the above metabolic pathways.This is the first time to elucidate the protective mechanism of Danshen on AD cell model by cellular metabolomics.2.Transmembrane transport of Danshen components on BBB cell model in vitroObjectives:To investigate the transporting mechanism of Danshen components across BBB,and clarify the relationship between Danshen components and transporters.Methods:BBB monolayer cell model was established by hBMEC and evaluated.The transmembrane transport experiment of appropriate concentration of tanshinone IIA,tanshinone I,dihydrotanshinone I,cryptotanshinone,caffeic acid,protocatechuic aldehyde and protocatechuic acid were carried out on BBB model.The quantitative determination method of Danshen components in HBSS was established by high performance liquid chromatography tandem triple quadrupole mass spectrometry.Results:The TEER of BBB cell model established by hBMEC was 33.04±5.02?·cm2,and the permeability coefficient of Na-F in the cell model was?3.42±0.47?×10-5 cm/s.Verapamil,a P-gp inhibitor,could inhibit the efflux and increase the absorption of Rhodamine 123 in the model.An HPLC-QQQ/MS-based quantitative method of Danshen components in HBSS was established.The specificity,matrix effect,accuracy and precision of the compounds met the standard.The Papp of tanshinone IIA,dihydrotanshinone I,cryptotanshinone,caffeic acid,protocatechuic aldehyde and protocatechuic acid were?3.757±1.723?×10-8 cm/s,?4.643±2.012?×10-6 cm/s,?4.977±1.587?×10-6 cm/s,?2.147±1.010?×10-5 cm/s,?1.516±0.179?×10-5 cm/s,?4.369±1.410?×10-5 cm/s,respectively.When the above compounds were combined with verapamil or Ko143 respectively,the efflux rate?ER?did not change significantly,and ERs were less than 2.Conclusion:Tanshinone IIA,dihydrotanshinone I,cryptotanshinone,caffeic acid,protocatechuic aldehyde and protocatechuic acid can be transported across BBB cell model,and were not effluxed by P-gp and BCRP.The transmembrane transport of these compounds in BBB cell model may be passive transcellular transport.3.Determination of Danshen components in rat plasma and brain tissue and study on transport across BBBObjective:To investigate the transport of Danshen components across BBB in rats and to elucidate the relationship between Danshen components and transporters.Methods:Quantitative methods of Danshen components in plasma and brain tissue were established by HPLC-QQQ/MS.The samples of plasma and brain tissue were prepared by liquid-liquid extraction.Gradient elution was performed at a flow rate of 0.400 mL/min.Quantitative analysis was carried out by double internal standard and multi-reaction monitoring mode was used for mass spectrometry.Thirty-six rats were randomly divided into control group and inhibitor group.Rats in inhibitor group were intraperitoneally injected with verapamil?40 mg/kg,1 mL/200 g?,while rats in control group were intraperitoneally injected with saline?1 mL/200 g?.After 60 minutes,all rats were given Danshen extract?25 g/kg,4mL/200 g?.At 30,60 and 90 minutes after the administration of Danshen extract,six of plasma samples and brain tissues of control groups and inhibitor groups were collected according to the order of administration,respectively.By determining the contents of Danshen components in the samples,the transport of Danshen components across BBB were analyzed,and the effects of P-gp inhibitor verapamil on the transport of Danshen components across BBB were evaluated.Results:The specificity of six components in plasma and brain tissue by HPLC-QQQ/MS were good.The RSD of intra-day and intra-day precision were less than 13.72%,the matrix effect was 72.49-138.29%,and the stability were satisfactory.Cryptotanshinone,dihydrotanshinone I,tanshinone I,caffeic acid and protocatechuic acid were detected in the plasma of the control group.Compared with the control group,the cryptotanshinone content in the plasma of the inhibitor group was higher,while that of dihydrotanshinone I,tanshinone I,caffeic acid and protocatechuic acid decreased.Only cryptotanshinone,dihydrotanshinone I and tanshinone I were detected in the brain tissue of rats in the control group and the inhibitor group.Compared with the control group,the contents of cryptotanshinone,dihydrotanshinone I and tanshinone I in the brain tissue decreased in the inhibitor group.Compared with the control group,the brain-blood ratio of cryptotanshinone,dihydrotanshinone I and tanshinone I decreased in the inhibitor group.Conclusion:Cryptotanshinone,dihydrotanshinone I and tanshinone I can be transported across the blood-brain barrier,while caffeic acid and protocatechuic acid can not be transported across BBB to brain tissue.In conclusion,the protective effect of Danshen on AD model cell was further verified to illustrate its clinical therapeutic effect.Tanshinone IIA,dihydrotanshinone I,cryptotanshinone,caffeic acid,protocatechuic aldehyde and protocatechuic acid can be transported across BBB cell models,and not affected by P-gp and BCRP inhibitors.In vivo,only cryptotanshinone,dihydrotanshinone I and tanshinone I can transport across the BBB.Although previous study suggest that cryptotanshinone is the substrate of P-gp,this study found that verapamil can reduce the brain-blood ratio of cryptotanshinone and dihydrotanshinone I.Dihydrotanshinone I,tanshinone I,caffeic acid and protocatechuic acid may not be the substrates of P-gp. |
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