ABSTRACT
Objective N-acety-L-cysteine (NAC) can attenuate the injury of podocytes and renal tubular epithelial HK-2 cells induced by contrast agents, but its specific action mechanisms needs to be further clarified. In this study, we investigated the effects of NAC on iopromide (IPM)-induced injury and the NF-κB/NLRP3 signaling pathway in HK-2 cells. Methods Renal tubular epithelial HK-2 cells were divided into seven groups, control, IPM, and IPM + NAC at 2, 4, 8, 16 and 32 mmol/L. After a 24-hour treatment of the HK-2 cells with NAC, CCK-8, DAPI staining, DCFH-DA and Western blot were employed for determination of the viability, apoptosis and morphology of the cells as well as the level of reactive oxygen species (ROS) and the expressions of Bax, Bcl-2, NLRP3, ASC, Caspase-1, IL-1β and NF-κB in the cells. Results Compared with the control, the cells of the IPM group showed a significantly reduced viability ([100 ± 4.749]% vs [48.819 ± 2.045]%, P < 0.05), increased apoptosis, elevated ROS level, and up-regulated expressions of Bax, Bcl-2, NLRP3, ASC, Caspase-1, IL-1β and NF-κB. In comparison with the IPM group, the HK-2 cells treated with NAC at 2, 4, 8, 16 and 32 mmol/L exhibited a remarkably increased viability ([55.398 ± 3.609]%, [58.953 ± 2.859]%, [61.531 ± 5.179]%, [59.845 ± 6.365]% and [59.094 ± 6.285]%) and decreased ROS level and expressions of Bax, Bcl-2, NLRP3, ASC, Caspase-1, IL-1β and NF-κB. The mean fluorescence intensity was significantly higher in the HK-2 cells of the IPM group than in the control cells (5050.85 ± 606.76 vs 1502.17 ± 55.91, P < 0.05), but remarkably decreased in those treated with NAC at 2, 4, 8, 16 and 32 mmol/L (4065.39 ± 106.59, 4162.05 ± 28.93, 3675.71 ± 50.38, 3133.79 ± 66.07 and 2675.80 ± 92.39) (P < 0.05). Conclusion NAC can effectively improve IPM-induced injury of renal tubular epithelial cells, which may be associated with its abilities of inhibiting ROS production and activating the NF-κB/NLRP3 signaling pathway.
ABSTRACT
Objective: To investigate the effect and mechanism of salvianolic acid B on the apoptosis of human renal proximal tubular epithelial cells (HK-2) induced by iopromide. Methods HK-2 cells were divided into eight groups: control group, model group, different concentrations of salvianolic acid B (1, 10, 50, 100, and 200 μmol/L) treatment groups and salvianolic acid B control group (200 μmol/L). The effect of salvianolic acid B on the proliferation of HK-2 cells induced by iopromide was detected by CCK-8 method. The changes of nuclear morphology were observed by DAPI staining. Levels of ROS in different groups were observed by fluorescence microscope and flow cytometry. The expression levels of Bax, Bcl-2, cleaved Caspase-3, p-Akt, p-ERK1/2, and Klotho were detected by Western blotting. Results:Compared with control group, the cell viability of HK-2 cells in model group was decreased significantly (P < 0.01), some nucleus appeared apoptotic characteristics such as chromatin condensation and nuclear division, the level of ROS and the expression of Bax, cleaved Caspase-3, p-ERK1/2 were increased significantly (P < 0.05, 0.01); Meanwhile, the expression of Bcl-2, p-Akt, and Klotho were decreased remarkably(P < 0.05, 0.01). However, the above effects of iopromide can be partially reversed by salvianolic acid B at 50, 100, and 200 μmol/L. But low concentration of salvianolic acid B (1 and 10 μmol/L) showed no obvious protective effect on the injury of HK-2 cells induced by iopromide. Conclusion: Salvianolic acid B can inhibit the apoptosis of HK-2 cells induced by iopromide, the mechanism may be related to anti-oxidative stress, activation of Akt, inhibition of ERK pathway and up-regulation of Klotho expression.
ABSTRACT
ObjectiveIopromide can induce injury to HK-2 cells, but its exact mechanism remains poorly understood. This study aimed to explore the influence of iopromide on ROS-NLRP3 inflammasome signaling in HK-2 cells.MethodsHK-2 human renal tubular epithelial cells were divided into six groups: control and iopromide at 37, 74, 111, 148 and 185 mgI/mL. The HK-2 cells in the latter five groups were treated with different concentrations of iopromide for 24 hours. Then the ROS level in the cells was detected by 2′,7′-Dichlorodihydrofluorescein diacetate staining and flow cytometry and the protein expressions of NLRP3, ASC, caspase-1, IL-1β, NF-κB and TNF-α determined by Western blot.ResultsThe ROS level was significantly increased in the HK-2 cells treated with iopromide at 37 mgI/ml (4103.89±98.89), 74 mgI/mL (4450.12±108.90), 111 mgI/mL (5050.85±606.76), 148 mgI/mL (6210.57±145.74) and 185 mgI/ml (7105.13±426.63) as compared with that in the control group (2551.71±84.00) (P<0.05). Western blot showed markedly upregulated expressions of NLRP3, ASC, caspase-1, IL-1β and TNF-α in the HK-2 cells in all the latter five groups in comparison with the control (P<0.05) and an increased level of NF-κB after treated with iopromide at ≥111 mgI/ml (P<0.05).ConclusionIopromide may induce injury to HK-2 cells by activating the ROS-NLRP3 inflammasome signaling pathway.