The Inhibitory Effect Of Taxifolin On Cell Damage And Adhesion Induced By Cr(Ⅵ)

Hexavalent chromium[Cr(VI)]is an important environmental pollutant.Cr(VI)is mainly derived from leather preparations,chrome plating of metal parts,industrial pigments and so on.Cr(VI)could enter the bloodstream by breath,diet and skin contact.Clinical data suggest that cardiovascular diseases(CVDs)were closely related to heavy metal contamination.Its possible mechanism of action is to cause oxidative stress in vascular endothelial cells,lead to the release of toxic free radicals,which may eventually lead to the occurrence of CVDs.Flavonoids were widely found in plants,and have important medicinal properties.What more,these compounds could improve the permeability of blood vessels,lower blood fat and cholesterol,and thus prevent their cardiovascular diseases.Taxifolin was a natural flavonoid with significant antioxidant activity,and could effectively inhibit cell damage caused by oxidative stress and other factors.At the same time,Taxifolin was not easily soluble in water,and carrier transporter protein can play an effective role.Human serum albumin(HSA)is an important transporter protein in the human body that binds to many endogenous and exogenous substances such as metabolites,drugs and other biologically active compounds for storage and transport.Therefore,studies on the interaction of small molecules such as Taxifolin with HSA are useful for understanding the distribution,transport,and metabolism of small molecules.Firstly,the inhibition of HUVEC cells growth by Cr(Ⅵ)at different concentrations(10,20,40,60,80,100,150 and 200 μ[M)were detected by MTT assay,and the inhibition of THP-1 cells growth by Cr(Ⅵ)at different concentrations(1,2,3,4,5,6 and 7 μM)were detected by CCK-8 assay.The oxidative stress level of HUVEC cells were detected by DCFH-DA fluorescent probe assay.The effect of different concentrations of Cr(Ⅵ)on apoptosis of HUVEC cells were detected by Hoechst 33258 assay.The effect of Cr(Ⅵ)(20μM)on the mRNA transcription level of related inflammatory factors in HUVECs were detected by Real-Time PCR.The effect of Cr(Ⅵ)on p38 MAPK and JNK signaling pathways and mitochondrial apoptosis signaling pathway in HUVEC cells were studied by Western blot assay.The results showed that 10 μM and 1 μM Cr(Ⅵ)could significantly inhibit the proliferation of HUVEC and THP-1 cells,10 μM Cr(Ⅵ)could enhance the oxidative stress in HUVEC cells and induce apoptosis of HUVEC cells,and there was a certain dose-effect relationship,inflammatory non-specific inflammatory markers C-reactive protein(CRP),tumor necrosis factor-α(TNF-α),inflammatory granule regulatory gene NLRP3,and cells were stimulated by HUVEC cells after Cr(Ⅵ)treatment Activation of the cytokine ICAM-1 involved in the inflammatory response and up-regulation of the related cytokines IL-1β and IL-6 involved in the inflammatory response in the interleukin family.It were activated that the p38 MAPK and INK signaling pathways in HUVEC cells by Cr(Ⅵ).Up-regulation of the pro-apoptotic protein Bax,while inhibiting the expression of the apoptotic protein Bcl-2.At the same time,it were activated that the NF-κB,p38MAPK and JNK signaling pathway in THP-1 cells by Cr(Ⅵ).Secondly,the cells were pretreated with Taxifolin to detect the inhibitory effect of Taxifolin on Cr(Ⅵ)-induced cell injury.The MTT and CCK-8 assays were used to detect the induction of HUVEC and THP cells by Cr(Ⅵ).The protective effect of cell viability was reduced,the ROS fluorescent probe assay was used to detect the change of ROS production induced by Cr(Ⅵ)in HUVEC cells by Western blot,the p38 MAPK,JNK and Nrf2 signals in HUVEC cells were detected by Western blot.Pathway and mitochondria influence the expression of apoptotic signals in vivo.The effect of Taxifolin on the expression of NF-κB,p38MAPK and JNK signaling pathway in THP-1 cells was examined.The results showed that Taxifolin inhibited the activity of HUVEC and THP-1 cells induced by Cr(Ⅵ).The results of ROS fluorescence probe showed that Taxifolin inhibited the production of ROS in HUVEC cells induced by Cr(Ⅵ).The results of Western blot showed that Taxifolin Inhibition of activation of p38 MAPK and JNK signal pathways in HUVEC cells and detoxification by activation of the Nrf2 signaling pathway.At the same time,taxifolin inhibited the activation of NF-κB signaling pathway in THP-1 cells induced by Cr(Ⅵ).Finally,the effects of the action of Taxifolin and HSA on the structure and binding mode of HSA were studied by spectroscopic method and molecular docking method,so as to explore the possible mechanism of in vivo transportation of Taxifolin The results showed that the hydrophobic group of tryptophan and tyrosine residues inside the molecule were exposed while the addition of Taxifolin,and the HSA peptide chain were stretched.The ultraviolet spectrum showed obvious color enhancement effect and the secondary structure change of HSA molecule.The mechanism of extinction is static quenching,The main force of interaction between Taxifolin and HSA is hydrogen bond calculated by thermodynamic formula,the tyrosine residue microdomain and tryptophan in HSA were determined by synchronous fluorescence spectroscopy.The effect of residue microdomains indicates that Taxifolin can alter the microenvironment around tyrosine and tryptophan in HSA,molecular docking results indicate that the most likely binding site of Taxifolin is a point in HSA.The results showed that Cr(Ⅵ)could induce HUVEC cells injury,activate p38 MAPK and JNK signaling pathway,and promote the release of related inflammatory factors(IL-1β,IL-6,TNF-α)and adhesion factors.At the same time,Cr(Ⅵ)could induce THP-1 cell damage,activate NF-κB,p38MAPK and JNK signaling pathway,and release IL-1β into the extracellular domain.Taxifolin could inhibit the oxidative stress induced by Cr(Ⅵ)on HUVEC cells and inhibit the adhesion reaction.At the same time,Taxifolin could inhibit Cr(Ⅵ)activation of NF-κB,p38MAPK and JNK signaling pathway in THP-1 cells and protect against inflammatory reactions.The above results indicate that Taxifolin has a good protective effect on two different types of cells induced by Cr(Ⅵ),and provides theoretical and experimental basis for the occurrence and prevention of CVDs caused by Cr(Ⅵ).