Study On The Mechanism Of Active Components Of Radix Paeoniae Alba Regulating P-gp-mediated Alkaloids Transport Across Blood Brain Barrier

Radix paeoniae alba is the dried root of Radix Paeoniae Alba Pall.,a perennial herb in the Ranunculaceae family.It has the functions of softening the liver and relieving pain,restraining yin and antiperspiration.There are hundreds of ancient prescriptions containing Radix paeoniae alba medicinal materials,such as Huangqin Decoction,Guizhi Decoction,Zhigancao Decoction and so on.At present,more than140 chemical components have been isolated from RPA,mainly monoterpene glycosides,triterpenoids,tannins,flavonoids,etc.Terpenes and terpene glycosides are the main active components of Radix Paeoniae Alba.Among them,paeoniflorin is the one with the highest content of monoterpenoids,accounting for about 90% of the total amount of paeoniflorin.Next is the liposide component,such as albiflorin,which is a kind of terpene glycoside compound.Studies have shown that Radix paeoniae alba has the effects of neuroprotection,anti-inflammatory,liver protection,analgesia,and nourishment of blood.The neuroprotective effect of Radix Paeoniae Alba and its active ingredients has been a hot research topic in recent years.In vivo and in vitro experimental studies have shown that paeoniflorin and albiflorin can penetrate the blood-brain barrier,are substrates of the transporter P-gp,and can mediate the efflux of central toxic alkaloids.The previous MDCK-MDR1 cell model transmembrane transport study in this laboratory showed that strychnine/ brucine is compatible with paeoniflorin or albiflorin,and compared with alkaloids(strychnine/brucine)alone,the efflux of alkaloids improved,the expression of P-gp increased,and the toxicity attenuated.This study will explore the interaction between the active ingredients of Radix Paeoniae Alba and the neurotoxicity targets of alkaloids through network pharmacology and molecular docking technology on the basis of preliminary laboratory performances,and investigate the compatibility of paeoniflorin with alkaloids(aconitine,hypaoconitine,etc.).The protective effect of aconitine,hypoaconitine and strychnine)on neurotoxicity in rats,further study the active components of Radix paeoniae alba(paeoniflorin,albiflorin)and alkaloid components(aconitine,strychnine,hypoaconitine and peimine),The effect of transmembrane transport and Pgp protein expression,to investigate the active components of Radix Paeoniae Alba(paeoniflorin,albiflorin)and alkaloids(brucine,strychnine,aconitine,hypoaconitine and peimine)on ATPase activity,P-gp functional activity,MDR1 m RNA expression level.Research methods1.In this study,an open field experiment was used to study the overall effect of the active ingredients of Radix Paeoniae Alba on the neurotoxicity of alkaloids in rats.The HE staining method was used to detect the microscopic changes of rat cerebral cortex morphology,and the ELISA method was used to detect tryptophan hydroxylase(TPH),tyrosine hydroxylase(TH),brain-derived nerves Nutritional factor(brainderived neurotrophic factor,BDNF)and nerve growth factor(Nerve growth factor,NGF)content in rat serum and tissue(prefrontal cortex,hippocampus,striatum,cerebellum and hypothalamus).2.Screen the neuroprotective active ingredients and targets of Radix Paeoniae Alba through network pharmacology,find neurotoxicity target proteins,match the targets of the active ingredients and neurotoxicity targets of Radix Paeoniae Alba,and perform PPI network interaction on the intersection proteins Analysis,GO biological pathway enrichment and KEGG signal pathway enrichment,establish a componenttarget-pathway network relationship diagram,and use molecular docking technology to verify component-target matching results.3.Using sybyl molecular docking software,use Radix paeoniae alba ingredients(paeoniflorin,albiflorin,gallic acid)and alkaloid ingredients(strychnine,brucine,aconitine,hypoaconitine,peimine)docking with the key proteins P-gp,MRP1,Claudin,and ZO-1,which are the key proteins of drug penetrating blood-brain barrier,to study the binding mode of each drug component and target protein,and evaluate its binding ability.4.Using the MTT method to determine the toxicity of alkaloids(aconitine,hypoaconitine,peimine)and Radix paeoniae alba components(paeoniflorin,albiflorin)and their compatibility on MDCK-MDR1 cells.To study the effect of the active ingredients of Radix Paeoniae Alba on the cross-MDCK-MDR1 blood-brain barrier of alkaloids.5.Western blot technology was used to detect the effect of alkaloids and active ingredients of Radix Paeoniae Alba and their compatibility on the expression of P-gp on the blood-brain barrier model of MDCK-MDR1 cells.6.Use the Pgp-Glo TM detection system to detect the effects of alkaloids,active ingredients of Radix Paeoniae Alba and their compatibility on ATPase activity on MDCK-MDR1 cell blood-brain barrier model.7.Use flow cytometry to study the effects of alkaloids,active ingredients of Radix Paeoniae Alba and their compatibility on the uptake of the probe drug Rhodamine 123(Rh123)on MDCK-MDR1 cell blood-brain barrier model,and explore alkaloids if it have any effect on the efflux function of P-glycoprotein with the active ingredients of Radix Paeoniae Alba and the compatibility of the two.8.Using RT-PCR method to investigate the effect of alkaloids,active ingredients of Radix Paeoniae Alba and their compatibility on the expression of MDR1 m RNA on the blood-brain barrier model of MDCK-MDR1 cells.Results1.Research on the protective effect of alkaloids and active ingredients of Radix paeoniae alba on the central nervous system of rats1.1 Results of the open field experiment:Compared with the blank control group,the aconitine group,hypoaconitine group,strychnine group and the positive drug scopolamine group have extremely significant differences in the incubation period,the number of grids,the peripheral time and the number of standing(P<0.01),manifested as prolonged incubation period,decreased number of spans,increased peripheral time,and decreased number of standings.It shows that aconitine,hypoaconitine,strychnine and the positive drug scopolamine have inhibitory effects on the central nervous system.Compared with the alkaloid single-use group,the aconitine-paeoniflorin compatibility group,the hypoaconitine-paeoniflorin compatibility group,and the strychnine-paeoniflorin compatibility group have extremely significant(P<0.01)incubation period,number of grids,peripheral time and standing times,manifested as a decrease in the incubation period,an increase in the number of grids,a decrease in peripheral time,and an increase in the number of standings,indicating that paeoniflorin significantly reduces the inhibition of aconitine,hypoaconitine and strychnine on the central nervous system effect.1.2 HE staining experiment results: compared with the blank group,the alkaloid group(aconitine group,hypoaconitine group,strychnine group)cerebral cortex nucleolus area atrophy,cytoplasmic vacuolation phenomenon,neuron damage.Compared with the alkaloid group,the aconitine-paeoniflorin,hypoaconitine-paeoniflorin and strychnine-paeoniflorin compatibility group significantly reduced the neuron damage in the cerebral cortex.The aconitine group,hypoaconitine group,strychnine group and the positive drug scopolamine group have all in the incubation period,the number of grids,the peripheral time and the number of standings.1.3 ELISA test results: the alkaloid single-use group(aconitine,hypoaconitine and strychnine)rat prefrontal cortex,hippocampus,striatum,cerebellum,hypothalamus and serum for 7,11,and 15 days.The expression of TPH,TH,BDNF,and NGF decreased.The expression of TPH,TH,BDNF,and NGF in the prefrontal cortex,hippocampus,striatum,cerebellar hypothalamus and serum of aconitine,hypoaconitine and strychnine after compatibility with paeoniflorin,respectively,for 7,11,and 15 days.The significant increase indicates that paeoniflorin can significantly improve the toxic damage of the central nervous system in rats caused by aconitine,hypoaconitine and strychnine.2.Results of network pharmacological research on the neuroprotective active components of Radix Paeoniae Alba and neurotoxicity targetsIn this chapter,through network pharmacology and literature search,a total of 23 active ingredients of Radix Paeoniae Alba were screened,and 190 intersecting targets with neurotoxicity prediction targets were screened.The top 20 targets were further screened,namely AKT1 and GAPDH,IL6,VEGFA,MAPK3,TNF,SRC,EGFR,MAPK1,HRAS,PTGS2,CCND1,STAT3,ESR1,HSP90A1,MMP9,APP,FGF2,BCL2L1 and AR,these targets are more common in protein interaction networks Protein is an important node that is related and embodies the core role.These targets involve a total of 216 GO biological processes and 151 KEGG signaling pathways.The molecular docking scoring of the active ingredients of Radix Paeoniae Alba and their targets showed that the active ingredients and their targets had a good interaction.3.Docking results of alkaloids and active ingredients of Radix Paeoniae Alba with P-gp,MRP1,Claudin,and ZO-1 protein moleculesThe results of molecular docking showed that the active ingredients of Radix Paeoniae Alba(paeoniflorin,albiflorin,gallic acid)and alkaloids(brucine,strychnine,aconitine,hypoaconitine,peimine)can interact with P-gp,MRP1,Claudin,and ZO-1proteins through the amino acid residue binding sites to produce hydrogen bond interactions,reflecting the characteristics of these components were substrates of target proteins.4.Results of cytotoxicity and translocation across the blood-brain barrier of alkaloids,active ingredients of Radix Paeoniae Alba and their compatibilityThe cytosafe toxicity range of each drug is: aconitine 0~80μg/m L,hypoaconitine0~50μg/m L,peimine 0~50μg/m L,paeoniflorin 0~1200μg/m L,albiflorin 0~1200μg/m L.And carried out the toxicity experiment of the compatibility of the alkaloids and the Radix Paeoniae Alba ingredients in various proportions.After the compatibility,the toxicity range of the alkaloids can be expanded,the best compatibility ratio can be screened out,and the concentration and ratio can be determined for the following experiments.The best compatibility concentration and ratio screened are:aconitine 80μg/m L-paeoniflorin 80μg/m L(1:1),aconitine 80μg/m L-albiflorin80μg/m L(1:1),hypoaconitine 50μg/m L-paeoniflorin 100μg/m L(1:2),hypoaconitine50μg/m L-albiflorin 100μg/m L(1:2),Peimine 50μg/m L-paeoniflorin 100μg /m L(1:2),peimine 50 μg/m L-albiflorin 50 μg/m L(1:1).Aconitine,hypoaconitine and peimine are well absorbed in MDCK-MDR1 cells,and the transport mechanism is passive transport;paeoniflorin and albiflorin are moderately absorbed and also exhibit passive transport.Paeoniflorin,albiflorin,aconitine,hypoaconitine and peimine are all affected by the efflux of P-gp protein.The active components of Radix Paeoniae Alba are combined with alkaloids,which can significantly promote the exudation of alkaloids.row.5.The effect of alkaloid components and active ingredients of Radix Paeoniae Alba on P-gp expression in MDCK-MDR1 cellsCompared with the blank group,the active ingredients of Radix Paeoniae Alba(paeoniflorin and albiflorin)alone had a significant down-regulation effect on the expression of P-gp.In the alkaloid group,indicating that hypoaconitine down-regulated the expression of P-gp,neither aconitine nor peimine had a significant effect on the expression of P-gp;compared with the alkaloid single-use group,each compatibility group can significantly increase the expression of P-gp.7.The effect of alkaloids and active ingredients of Radix Paeoniae Alba on the function of P-gpCompared with the blank group,the fluorescence intensity of paeoniflorin,albiflorin,brucine,strychnine and each compatible group increased significantly,indicating that the intracellular the accumulation of Rh123 increased,indicating that the efflux function of P-gp was inhibited,aconitine,hypoaconitine,peimine,had no difference.Compared with the alkaloid single-use group,the fluorescence intensity of the compatibility group was significantly reduced,reflecting that the compatibility enhances the function of P-gp and increases the efflux of Rh123 by P-gp.8.The effect of alkaloids and active ingredients of Radix Paeoniae Alba on the expression of MDR1 m RNACompared with the blank group,paeoniflorin,albiflorin,peimine,and strychnine can significantly increase the expression of MDR1 m RNA in MDCK-MDR1 cells,there was no significant difference between aconitine,hypoaconitine and the blank group,strychnine can significantly reduce the expression of MDR1 m RNA.Compared with the alkaloid single-use group,each compatibility group can significantly increase the expression of MDR1 m RNA.ConclusionThis paper studies the reduction of alkaloid components and active ingredients of Radix Paeoniae Alba from the perspectives of network pharmacology,molecular docking,rat neurotoxicity test,P-gp expression,P-gp ATPase,P-gp function,and m RNA expression.The toxicity mechanism provides a new perspective for the study of the attenuation mechanism of neurotoxic alkaloids,and also expands the application space of Radix Paeoniae Alba with neurotoxic alkaloids,providing data support for clinical applications.