Salvianolic Acid B Attenuates Iopromide-induced Renal Tubular Epithelial Cell Injury By Inhibiting The TLR4/NF-κB/NLRP3 Signaling Pathway

Objective: The study was conducted to investigate whether Sal B alleviates iopromideinduced injury of human proximal renal tubular epithelial cells by inhibiting TLR4/NF-κB/NLRP3 signaling pathway.Methods: HK-2 cells were treated with several concentrations of iopromide(50,100,150,200 mg I/m L)for 3 h,the cell viability was detected by Cell Counting Kit-8(CCK-8)assay and finally 150 mg I/m L iopromide was used to induce HK-2 cells to establish an in vitro damage model of post-contrast acute kidney injury(PC-AKI).After the HK-2 cells were pretreated with different concentrations of Sal B(10,50,100 μmol/L),the cell viability was measured by CCK-8 assay.The levels of reactive oxygen species(ROS)and the mitochondrial membrane potential(△Ψm)were evaluated by DCFH-DA and rhodamine123(Rh123)staining,separately.The average fluorescence intensity was measured by flow cytometry.The nucleus morphology of apoptotic cells was observed by DAPI staining.The protein expression levels of TLR4,NLRP3,ASC,Caspase-1,p-NF-κB,IL-18,IL-1β,TNF-α.cleaved caspase-3,and the ration of Bax/Bcl-2 were detected by Western blot.The free energy of docking between the ligand(Sal B)and the target proteins(TLR4,NLRP3)was calculated by the Molecular Operating Environment(MOE)software,and the docking amino acid residues were analyzed.In addition,TLR4 inhibitor(TAK-242)was added to pretreat cells,and cell viability,ROS,MMP,apoptosis rate,and the expression levels of the above proteins were detected.Results:1.Iopromide induced HK-2 cell injury by activating TLR4/NF-κB/NLRP3.Compared with the control group,the cell viability of the iopromide group was significantly decreased,the level of ROS was significantly increased while the level of △Ψm was decreased.The number of apoptotic cells,the expression levels of cleaved caspase-3 and the ratio of Bax/Bcl-2 were significantly increased in iopromide group.The expression levels of TLR4,NLRP3,ASC,Caspase-1,p-NF-κB,IL-18,IL-1β,TNF-α were significantly increased in iopromide group.2.Sal B alleviated the inflammatory injury of HK-2 cells induced by iopromide.Compared with the iopromide group,the cell viability in 100 μmol/L Sal B group was significantly decreased,the level of ROS was significantly increased while the level of △Ψm was decreased.The number of apoptotic cells and the expression levels of above-mentioned proteins were decreased.The effect of 5 μmol/L TAK-242 was similar to that of 100 μmol/L Sal B.However,the pre-treatment group with TAK-242 and Sal B at the same time had no significant difference in cell viability and apoptosis rate compared with the pre-treatment group with TAK-242 or Sal B alone.Conclusion:1.Iopromide may induce HK-2 cell injury by activating TLR4/NF-κB/NLRP3 signaling pathway.2.Sal B attenuated iopromide-induced HK-2 cell injury,reduced apoptosis and ROS generation,enhanced ΔΨm level,and inhibited TLR4/NF-κB/NLRP3 signaling pathway.