The Effects Of β-casein And Lactoferrin On The Damage And Hypoglycemic Activity Of Vanadate

Diabetes mellitus is a chronic metabolic disease in global terms.Long-term high levels of blood sugar in patients can cause damage to various organs and tissues,and even lead to premature death in severe cases.As a trace element contained in the human body,vanadium has various physiological effects,such as anti-inflammatory and anti-cancer effects,etc.Meanwhile,vanadium has an"insulin-like effect",which can promote glucose homeostasis and restore insulin sensitivity,and has a good therapeutic effect.However,the inorganic vanadium salt has certain biological toxicity and low bioavailability,which limits its application in the treatment of diabetes.Studies have shown that the toxicity of vanadate can be alleviated when used with polyphenols or hypoglycemic agents.However,the application of vanadate with natural proteins is rarely reported.In this work,we investigated the interaction mechanism of vanadate withβ-casein and lactoferrin,as well as the effects of these two proteins on the cytotoxicity and hypoglycemic activity of vanadate.Our research provided a new idea and experimental basis for the application of vanadate compounds in the treatment of diabetes.In this study,the interaction mechanism of vanadate withβ-casein and lactoferrin was investigated,and the intrinsic fluorescence quenching mechanism of vanadate for the two proteins was explored through fluorescence spectra.The binding sites and the types of binding forces were explored according to thermodynamic analysis.The effects of vanadate on the conformation ofβ-casein and lactoferrin were investigated by ultraviolet spectroscopy（UV-Vis）,synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,circular dichroism spectroscopy（CD）and fourier transform infrared spectroscopy（FT-IR）.The binding sites,types of binding forces of vanadate withβ-casein and lactoferrin were investigated by molecular simulation.Meanwhile,the vanadate-protein complexes were prepared with the concentration ratio of 1:1,the inhibitory effects ofβ-casein and lactoferrin on vanadate cytotoxicity and the hypoglycemic activity of vanadate were studied.The effects of vanadate on survival rate of human embryonic kidney293（HEK293）cells treated with two proteins were detected by MTT assay.Cell apoptosis was detected by Hoechst33258 assay.Reactive oxygen species（ROS）fluorescence probe assay was used to detect the changes of intracellular reactive oxygen species expression.Rhodamine 123 kit was used to detect the changes of mitochondrial membrane potential.Western blot was used to detect the expression of mitogen activated protein kinase（MAPK）and apoptotic signaling pathway related proteins.Finally,insulin resistance model was established in human hepatocarcinoma（Hep G2）cells,and the effects ofβ-casein and lactoferrin on the hypoglycemic activity of vanadate were investigated by glucose oxidase assay.The results showed that vanadate could quench the intrinsic fluorescence ofβ-casein and lactoferrin,and the quenching mechanism was static quenching.Further analysis suggested that there was only one binding site between vanadate and the two proteins,the binding constants were 5.57 and 1.85×10⁴ M^-1（298K）,the interaction force was a spontaneous reaction dominated by electrostatic force.UV-vis spectra showed that vanadate could combine with the two proteins to form a complex.Synchronous fluorescence spectra and three-dimensional fluorescence spectra showed that vanadate treatment would change the microenvironment of amino acid residues of the two proteins.The results of circular dichroism and fourier transform infrared spectroscopy demonstrated that the interaction of vanadate withβ-casein and lactoferrin could cause the change of secondary structure ofβ-casein and lactoferrin.The results of molecular docking showed that vanadate could interact with amino acids aroundβ-casein and lactoferrin,and the main type of force was hydrophobic force.Futhermore,the results showed thatβ-casein and lactoferrin treatment could inhibit vanadate cytotoxicity,improve cell survival rate,reduce vanadate-induced apoptosis,inhibit the expression of apoptotic protein Bax,and increase the expression level of Bcl-2.In addition,it could reduce the intracellular ROS expression,reduce the oxidative stress response in HEK293 cells,and inhibit the MAPK signaling pathway activated by vanadate stimulation.It could be speculated that the combination ofβ-casein and lactoferrin with vanadate could reduce kidney injury.The glucose uptake experiment showed that the application of vanadate withβ-casein and lactoferrin still had good hypoglycemic activity.These results indicated that vanadate could interact withβ-casein and lactoferrin to form a complex,the cytotoxicity of vanadate could be alleviated by regulating oxidative stress and apoptosis,and still had hypoglycemic effect.This study elucidated the interaction mechanism of vanadate withβ-casein and lactoferrin,and proposes a method to alleviate the cytotoxicity of vanadate,which provides a new idea for the application of vanadate in the treatment of diabetes.