Transport Mechanism Of Strychni Semen Active Components With Radix Paeoniae Alba Active Components And Their Effects On P-Glycoprotein Expression Based On MDCK-MDR1 Cell Model

Strychni Semen is the dry and mature seed of Strychnos nux-vomica L.Clinically used for the treatment of carbuncle,rheumatoid arthralgia,rheumatism,numbness,etc.Brucine and strychnine are the main active ingredients in the scorpion,and also have strong toxic side effects.The therapeutic dose of scutellaria is close to the toxic dose,and the therapeutic window is narrow.Brucine and strychnine enter the body through different routes and are widely distributed,Brucine and strychnine has a strong central nervous system excitatory effect,Prone to neurological and cardiac poisoning reactions have greatly limited the use of Strychnos,Therefore,the study of the attenuating mechanism of Strychni Semen is of great significance.The previous transdermal absorption results of our research group showed that the combination of total glucosides of paeony and total alkaloids from seed of Strychnos nux-vomica L can reduce the transmission of toxic components.Previous studies on the pharmacokinetics and brain distribution in vivo by transdermal administration of brucine/strychnine combined with paeoniflorin at normal and toxic doses in rats,Results showed that brucine/strychnine combined with paeoniflorin could inhibit the absorption of strychnine in brain and blood and promote the penetration of paeoniflorin to a certain extent after transdermal administration.These can partially verify or explain the rationality of their extract compatibility.The previous research on the attenuation mechanism of strychni semen and paeoniae radix alba compatibility based on the MDCK-MDR1 cell model,MTT test results showed that when the ratio of Strychni Semen extract and Radix Paeoniae Alba extract was1:6,the attenuation effect was better.Transport experiment showed that Strychni Semen extract compatible with Radix Paeoniae Alba extract,apparent permeability coefficient of AP-BL of brucine and strychnine decreased significantly,the efflux ratio increased significantly,the results showed that the toxicity of Strychni Semen extract could be reduced by the combination of Radix Paeoniae Alba extract.However,the compatibility of brucine/strychnine with paeoniflorin/albiflorin/gallic acid based on MDCK-MDR1 cell model has not been reported.This experiment will be based on the previous part of the study,Taking brucine,strychnine,paeoniflorin,paeoniflorin,paeoniflorin and gallic acid as the research object,further clarification the toxicity,transport mechanism and the effect on P-gp expression of brucine/strychnine and paeoniflorin/paeoniflorin/gallic acid were used alone or in combination with each otherIn this study,MTT method was used to determine the safety range of MDCK-MDR1 cell when brucine/strychnine and paeoniflorin/albiflorin/gallic acid were used alone or in combination with each other.and the best ratio of brucine/strychnine to paeoniflorin/gallic acid was determined.Taking P_app,C_CUM and ER as indicators,study on transport mechanism of brucine/strychnine and paeoniflorin/paeoniflorin/gallic acid and their compatibility in MDCK-MDR1 cell model.Western blot was used to study the effects of brucine/strychnine and paeoniflorin/paeoniflorin/gallic acid on the expression of P-gp.The attenuation mechanism of brucine/strychnine and paeoniflorin/paeoniflorin/gallic acid and their compatibility were further analyzed.The specific experimental results are as follows:1.Results showed brucine,strychnine,Paeoniflorion,albiflorin and Gallic acid were non-toxic concentration ranges of 0⁰.1?g/mL,0⁰.04?g/mL,0⁵0?g/mL,0⁵0?g/mL,0²5?g/mL,Furthermore,brucine plus paeoniflorin?1:2 w/w,BP?,brucine plus albiflorin?1:1 w/w,BA?,and brucine plus Gallic acid?1:2,w/w/w,BG?were not cytotoxic at concentration ranges of 0¹.0?g/mL.strychnine plus paeoniflorin?1:4,w/w,SP?,strychnine plus albiflorin?1:3 w/w,SA?,and strychnine plus Gallic acid?1:2,w/w/w,SG?were not cytotoxic at concentration ranges of 0⁰.4?g/mL.brucine and strychnine have obvious toxicity to MDCK-MDR1 cell in a dose-dependent relationship,cytotoxicity of brucine is lower than that of strychnine.Paeoniflorion,albiflorin,Gallic acid can reduce brucinea and strychnine cytotoxicity in MDCK-MDR1.2.The effects of brucine/strychnine and paeoniflorin/paeoniflorin/gallic acid on the transport of MDCK-MDR1 cells were studied.The results showed that paeoniflorin/paeoniflorin could reduce the absorption of brucine/strychnine,increased the efflux rate,reduced the accumulation of brucine and strychnine in cells,and then played a role in reducing toxicity.After compatibility,the apparent permeability coefficients of paeoniflorin/paeoniflorin in BL-AP direction changed correspondingly.3.Western blot was used to study the effects of brucine,strychnine,paeoniflorin,paeoniflorin and gallic acid and their compatibility on the expression of P-gp.combination of brucine,strychnine with paeoniflorin,paeoniflorin,gallic acid significantly enhanced the efflux of P-gp.Promote the efflux of brucine and strychnine,reduce the accumulation of brucine and strychnine in cells,and then play a role in reducing toxicity.Previous studies on the effect of compatibility of Strychni Semen extrac and Radix Paeoniae Alba extract on the expression of P-gp,The results showed that the expression of P-gp increased significantly after the compatibility of Strychni Semen extrac and Radix Paeoniae Alba extract.The expression of P-gp in this experiment was similar to that in previous experiments.The results of this study explained the attenuation mechanism of brucine,strychnine combined with paeoniflorin,paeoniflorin and gallic acid.Further elucidation of toxicity reduction mechanism of Strychni Semen combined with Radix Paeoniae Alba from the point of view of single component.It provides a new way of thinking for the study of the mechanism of reducing toxicity of S Strychni Semen.It also expands the application space of Radix Paeoniae Alba.It lays a foundation for the development of new drugs.