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
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.
ABSTRACT
Objective Methylglyoxal can cause the injury of human proximal tubular epithelial cell line(HK-2 cells),but the exact mechanism is still unclear. The present study aimed to explore the influence of oxidative stress and the expression of NLRP3 inflammasome in HK-2 cells induced by methylglyoxal. Methods HK-2 cells at logarithmic phase were divided into six groups:control group and 100,200,400,800,1600 μmol/L methylglyoxal groups (cells were cultured in 100,200,400,800,1600 μmol/L methylg-lyoxal concentration for 24 h). Superoxide dismutase(SOD)levels were assayed by thibabituric acid method. Release of lactate dehydro-genase(LDH)activity was detected by assay kit.ROS production was measured by DCFH-DA staining. The expression levels of NLRP3,caspase-1,IL-1β and NF-κB were evaluated by western blot. Results Compared with control group,different methylglyoxal concen-trations could enhance ROS level and LDH activity in HK-2 cells(P<0.05)and reduce SOD level significantly(P<0.05). The results of western blot showed the protein levels of NLRP3,caspase-1,IL-1β and NF-κB were significant up-regulated after the addition of methylglyoxal(P<0.05). Conclusion Methylglyoxal may induce the injury of HK-2 cells by oxidant stress and activating of NLRP3 inflammasome signaling.
ABSTRACT
Background It is widely appreciated that many animal cells rely on the mechanism of regulatory volume decrease (RVD) after swell under the hypotonic environment,which involved in some processes of cellular physiology.But the RVD of lens epithelial cells(LECs) still is being further researched.Objective Present study was to clarify the possible mechanisms and influencing factors in the RVD of LECs.Methods Human LECs line (HLE B-3)were cultured and passaged in DMEM/F12 containing 10％ fetal bovine serum(FBS),and before volume measurement,cells were stuck to the base of a perfusion chamber,Ringer solution osmolality was decreased from 15％Hypo to 45％ Hypo,and the cells stimulated by 45％ Hypo Ringer solution were used as the control group.Some experiments were performed in the presence of high extracellular K+ concentration,chloride or potassium channel inhibitor,experiments were also carried out in the nominal absence of Ca2+,Cl-or HCO-3 to test the effect of a decrease in intracellular concentration of these ions on the cell volume response.The volume changes of living cells were measured by lag-time microphotograph acquisition and analysis system (IPP6.0).Results Time course of cell volume change after hypotonic shock in HLE B-3 cells was observed.The cell swelling was followed by a gradual volume recovery,indicating the presence of RVD was influenced by the hypotonic stress.Under the stimulation of 45 ％Hypo Ringer solution,the rates of RVD were (59.1 ±7.8)％.RVD was correlated positively to the maximum cell volume (r =0.99,P＜0.05)in S shape,and RVD changes were sensitive to alter maximum cell volume in the range of 115％-135％.RVD reduced to (16.5 ± 1.6) ％,(14.7 ± 2.3) ％,respectively after acted by potassium channel inhibitor,TEA(10 mmol/L)and BaCl2(5 mmol/L)as well as chloride channel inhibitor,NPPB(100 μmol/L)and DIDS(100 μmol/L),with significant differences in comparison with the control group(all P＜0.01).RVD decreased by(5.8±1.6)％ and(2.7±0.8)％ in high concentration of K+ in extracellular fluid and the absence of Cl-(P＜0.01).RVD was significantly inhibited under the absence of Ca2+.When the 45％ Ringer solution was pH6.8,the process of RVD delayed.The rate of RVD in the first ten minutes was (0.86±0.24)％/min,showing a significant decline in comparison with (3.24±0.84) ％ / min of pH 7.4 (P ＜0.05).Conclusions HLE B-3 have RVD ability under the hypotonic stress stimulation.A certain intracellular Ca2+ concentration is the premise of RVD activation,and Cl efflux and K+ efflux are the key mechanism of RVD of HLE B-3.Acidic environment of hypotonic solution delays the occurrence of RVD.