Study On The Mechanism Of Bushen-Yizhi Formula In Enhancing The Bioavailability Of Osthole In The Treatment Of AD Based On The CYP3A4 Metabolic Pathway

China has become the country with the largest number of patients with Alzheimer’s disease(AD),and the incidence of AD is becoming more and more prevalent."The core pathogenesis of AD is deficiency of kidney essence and marrow,resulting in loss of marrow filling and loss of nourishment for the mind,resulting in mental impairment,memory loss and other dementia manifestations.In response to the pathological characteristics of AD,Lai Shilong,professor of Guangzhou University of Traditional Chinese Medicine,designed the Bushen-Yizhi Formula(BSYZ)in his long-term clinical practice,which is good at tonifying the kidney and filling the marrow.The results of the previous clinical studies have shown that this formula can improve the cognitive function of AD patients,and it was found through experiments that BSYZ can improve the bioavailability of its main active ingredient,Osthole(OST),indicating that the formulation is scientifically and reasonable.However,its intrinsic biological mechanism is still unclear,which limits the exploration of its modern pharmacological basis.ObjectiveThis study investigates the effect of BSYZ on the bioavailability of its main active ingredient,OST,in vitro and in vivo,to reveal the pharmacological basis and molecular mechanism of BSYZ to improve the bioavailability of OST for the treatment of AD,to verifies the effect of the active ingredient in BSYZ to inhibit CYP3A4 on the bioavailability of OST,to improve the bioavailability of OST and thus enhance the anti-AD efficacy of BSYZ,and to provide an experimental basis for the scientific and rational formulation of traditional Chinese medicine.MethodsA comprehensive simulation screening of potential CYP3A4 inhibitors in BSYZ was conducted through network pharmacology and molecular docking.The optimal CYP3 A4 inhibitor compounds in BSYZ were further classified and validated by pharmacological evaluation in vitro,and a method based on liquid chromatography-mass spectrometry(LC-MS/MS)was developed for the determination of OST content in BSYZ.The pharmacokinetic mechanism of the optimal CYP3A4 inhibitor compound in BSYZ was investigated using SD rats to clarify the synergistic relationship between the two compounds.Subsequently,APP/PS1 double transgenic AD mice were used to investigate the learning and memory ability of model animals through behavioral studies such as water maze,and brain histopathology related indices such as Nissler staining and thiosemicarbazone staining to investigate the effect of CYP3A4 inhibitor compounds in BSYZ that we have validated in vitro to enhance the bioavailability of OST for the treatment of AD and related mechanism studies.Finally,we constructed APPswe-SH-SY5Y cell model overexpressing Swedish mutant amyloid precursor protein(APPswe)by lentiviral vector to further validate the biological mechanisms related to the effective active components of anti-AD in BSYZ.ResultsThe 131 compounds were screened as potential CYP3A4 inhibitors from the BSYZ compound library by molecular docking simulations,including several types of compounds such as saponins,glycosides,anthraquinones,etc.And 21 of them were found to be the most likely to inhibit CYP3A4 based on their chemical structures and mechanisms related to CYP3A4 inhibition.Gomisin B was found to have the strongest inhibitory effect on CYP3A4 at the same 5μM dosing concentration as the positive drug,ketoconazole(Ket).Through pharmacokinetic experiments we found that the Cmax of OST(185.93±101.17,P<0.001)after coadministration of Gomisin B with OST and the Cmax of OST(132.42± 17.54,P<0.001)after coadministration of Ket with OST was significantly higher than that of the OST group alone(P<0.001),indicating that Gomisin B greatly increased the concentration of OST in the plasma of SD rats.We then selected APP/PS1 double transgenic mice to explore the biological mechanisms of OST,Gomisin B,and OST+Gomisin B for the treatment of AD.The quantitative analysis of OST content in the brain tissue of each group of mice by LC-MS/MS method revealed that the co-administration of Gomisin B and OST significantly increased the OST content in the brain tissue of mice.From the behavioral experiments,it was observed that in addition to OST group,OST+Gomisin B and BSYZ group,Gomisin B alone administration group also showed significant improvement in memory dysfunction in APP/PS1 double transgenic mice,so a more in-depth related mechanism study was conducted.The results showed that the neuroprotective effects of OST and Gomisin B on APP/PS1 double transgenic mice may be related to the effects of reducing Aβ levels,improving oxidative stress and anti-apoptosis,suggesting that OST and Gomisin B may improve cognitive memory dysfunction in APP/PS1 double transgenic mice through multiple targets,while the OST+Gomisin B group showed a significant improvement in reducing Aβ levels,improved oxidative stress and anti-apoptotic effects than the OST or Gomisin B group administered alone.Finally,an APPswe-SH-SY5Y cell model was successfully constructed by lentiviral vector,and the effects and related mechanisms of CYP3A4 inhibitor-Gomisin B in BSYZ screened in vitro for AD treatment were examined and validated previously.The results of the previous study in the APP/PS1 double transgenic mouse model were further validated in the in vitro APPswe-SH-SY5Y cell model.low,medium and high doses of Gomisin B(50 μM,100 μM and 200 μM)significantly reduced the expression of Aβ1-42 in APPswe-SH-SY5Y cells and inhibited the expression of β-site Medium and high doses of Gomisin B(100 μM and 200 μM)upregulated total superoxide dismutase(T-SOD)expression in APPswe-SH-SY5Y cell model,while low,medium and high doses(50 μM,100 μM and 200 μM)reduced malondialdehyde(MDA)and reactive oxygen species(ROS)production.(ROS)production.The expression of Bcl-2 and the expression of Bax and Caspase3 in APPswe-SH-SY5Y cell model were significantly increased by the medium and high doses of Gomisin B(100 μM and 200μM).This suggests that Gomisin B exerts its anti-AD effects through ameliorating oxidative stress,regulating apoptosis-related proteins,inhibiting CYP3A4 and thus increasing the bioavailability of OST.ConclusionThis study screened and found a multifunctional bioactive component-Gomisin B in BSYZ,it could improve learning memory dysfunction in APP/PS1 double transgenic mice by ameliorating oxidative stress,anti-neuronal apoptosis and reducing Aβdeposition,while Gomisin B could also inhibiting CYP3A4,the main metabolic enzyme of OST,to increase the drug concentration of OST in blood and brain tissue,thus contributing to better anti-AD efficacy of OST.This study provides an experimental basis for the anti-AD efficacy of BSYZ,and provides some research ideas to explore the potential law and the compounding mechanism of traditional Chinese medicine.