Study On The Mechanism Of Action Between Glucocorticoids And Bovine Serum Albumin:Effects Of Metabolites And Combination Drugs

With the widespread clinical application of chemotherapy,the human body has become more and more resistant to drugs.The study of the coexistence of drug metabolites and the mother,as well as the combination of drugs,is of great significance to clinical medication.Glucocorticoid drugs（GCs）not only have good therapeutic effect,but also have the advantage of low price.GCs is widely used in clinic and is one of the commonly used anti-inflammatory drugs at present.By studying the interaction between GCs and its metabolites and serum albumin（SA）at different temperatures,we can determine the mechanism of interaction between GCs and its metabolites and the influence of metabolites on the interaction between mother and SA when both mother and metabolites exist.In addition,the study on the interaction between coexisting drugs and SA also provides a reasonable basis for clinical drug combination.In this paper,the interactions mechanism between GC and bovine serum albumin（BSA）were studied by various spectral methods.In order to understand the behavior of cortisone acetate（CA）and the effect of CA and its metabolite hydrocortisone（HS）on the conformation change of bovine serum albumin（BSA）,the interaction mechanism between CA/HS and BSA was studied by various spectroscopic techniques.The fluorescence quenching experiment results show that the fluorescence quenching mechanism of BSA by CA/HS is static quenching.The binding constants of CA-BSA/HS-BSA are 2.04×10⁴L·mol^-1and 2.02×10³L·mol^-1,and the number of binding sites were 0.90 and 0.76.Thermodynamic parameters show that the binding force between CA/HS and BSA is mainly driven by hydrophobic force.The results of competitive experiments showed that CA/HS was bound in the hydrophobic cavity of BSA site II.The secondary structure of BSA was changed by CA/HS through UV-vis spectra,three-dimensional fluorescence spectra,infrared fluorescence spectra and synchronous fluorescence spectra.The presence of metabolites did not change the quenching mechanism,but weakened the binding affinity of CA to BSA and increased the binding distance,which may be attributed to the competitive binding between CA/HS and BSA.This study revealed the combined action of CA and its metabolites at the molecular level of BSA.The mechanism of action of prednisone acetate（PA）and its metabolite prednisolone（PS）in the binary system and the ternary system of BSA was studied.Use fluorescence spectroscopy,ultraviolet-visible absorption spectroscopy,three-dimensional fluorescence spectroscopy,and infrared spectroscopy to characterize the binding mode of PA/PS with BSA under separate and coexisting conditions,the binding constant,and the effect on the secondary structure of BSA.Fluorescence spectroscopy experiment results show that PA and PS are combined with BSA through van Van der Waals’force and hydrogen bonds.At a temperature of 298 K,the binding constants of PA and PS with BSA are 1.27×10⁴L·mol^-1and 8.01×10⁴L·mol^-1,the number of drug molecules bound to each protein is 1.02（PA）and 1.19（PS）.The conformation of BSA was changed by the decrease ofα-helix,PA decreased from 49.75%（free BSA）to 40.64%,PS decreased to 41.52%.In addition,the peak shifts of UV-vis absorption spectra and synchronous spectra indicate that BSA may act as its carrier protein when PA and its metabolite PS are delivered to the target tissue,and exert its effect on the conformation and amino acid microenvironment of BSA at the same time.The results of site competition showed that PA/PS was bound to site II of BSA subdomain.In the presence of PS,the binding constant increased to 5.63×10⁴L·mol^-1,and theα-helix of PA-BSA decreased to 48.07%,indicating that the presence of PS enhanced the binding ability of PA to BSA,thus reducing the concentration of free drug in plasma and increasing its efficacy.Various spectroscopic methods were used to study the effect of the combination of levamisole（LMS）and dexamethasone（DEX）on the concept of BSA.The results show that the fluorescence quenching between DEX/LMS and BSA is a static quenching process.Both have the same fluorescence quenching type for BSA and similar binding force types.By comparing the quenching constants and binding constants of the two drugs LMS and DEX,it is found that DEX has a larger quenching constant for BSA and a stronger binding ability with BSA.The distance is closer.However,when LMS is present,the binding constant of DEX-BSA decreases and the binding distance increases.DEX and LMS have the same binding site for BSA（site II）.At the same time,the results of UV-vis absorption spectrum,synchronous fluorescence spectrum and three-dimensional fluorescence spectrum showed that DEX further induced the microenvironment and conformational changes of BSA.The existence of LMS did not change the conformation and microenvironment of DEX-BSA.