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Objective To investigate the effect of Xuebijing on paraquat-induced oxidative stress and inflammation level in human kidney cell line-2 (HK-2) cells. Methods The HK-2 cells were routinely cultured and divided into blank control group, paraquat poisoning model group, Xuebijing injection pretreatment group and Xuebijing control group according to random number table method. The concentration of paraquat in HK-2 cells were measured by high performance liquid chromatography (HPLC); the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were measured by chemical colorimetry method; the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). Results With prolonged treatment, paraquat concentration gradually increased in HK-2 cells in the paraquat poisoning model group and the Xuebijing injection pretreatment group, reaching the peak value after treatment for 48 hours, moreover, the concentration of paraquat in HK-2 in the Xuebijing injection pretreatment group was significantly lower than that in the paraquat poisoning model group (mg/L: 4.26±0.20 vs. 5.77±0.18, P < 0.05). Compared with the blank control group, the contents of MDA, TNF-α and IL-6 of paraquat poisoning model group were obviously elevated [MDA (nmol/mg): 4.47±0.10 vs. 2.21±0.08, TNF-α (ng/L): 206.91±13.22 vs. 98.14±5.67, IL-6 (ng/L): 253.33±5.22 vs. 116.97±13.54, all P <0.05], and the activity of SOD was significantly reduced (U/mg: 33.30±0.62 vs. 41.58±0.17, P < 0.05). Compared with paraquat poisoning model group, the contents of MDA, TNF-α and IL-6 in Xuebijing injection pretreatment group were obviously decreased [MDA (nmol/mg): 2.92±0.17 vs. 4.47±0.10, TNF-α (ng/L): 166.29±15.47 vs. 206.91±13.22, IL-6 (ng/L): 209.39±3.18 vs. 253.33±5.22, all P < 0.05], and the activity of SOD was significantly increased (U/mg:38.10±0.67 vs. 33.30±0.62, P < 0.05) in the paraquat pretreatment group. Conclusion Xuebijing could reduce the concentration of paraquat in HK-2 cells, and decrease oxidative stress and inflammation factor levels in HK-2 cells.
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Objective@#To investigate the protective effect and mechanism of Xuebijing (XBJ) on paraquat (PQ) -induced apoptosis in Human kidney cell line-2 (HK-2) cells.@*Methods@#Routinely cultured HK-2 cells, (1) Cell growth inhibition experiment after PQ and XBJ intervention: PQ was divided into 0、200、400、800、1600 and 3200 μmol/L PQ groups, and the cell survival rate was detected after intervening 24、48 and 72 h. XBJ was divided into 0、5、10、20、40 mg/ml XBJ groups, and the cell survival rate was detected after intervening 24、48 and 72 h.To determine the rational drug concentration and the duration of action of XBJ and PQ. (2) PQ-induced HK-2 cell growth inhibition experiment antagonized by XBJ: The cells were divided into normal control group, PQ group (800 μmol/L) and PQ+XBJ group (The cells were pretreated with 5、10 and 20 mg/ml XBJ for 1 h, then cultured with PQ of 800 μmol/L) , After cultured 24 h、48 h and 72 h separately, the cell survival rate was detected. (3) HK-2 cells were divided into normal control group、PQ group (800 μmol/L PQ cultured for 24 h) 、PQ+XBJ group (pretreated with 10 mg/ml XBJ for 1 h, and then 800 μmol/L PQ cultured for 24 h) and XBJ group (10 mg/ml XBJ cultured 24 h). The apoptosis of cells was detected by flow cytometry. The protein expression of Bcl-2 and BAX in each group was detected by Western blotting. The expressions of caspase-3 and caspase-9 were detected by caspase-3 and caspase-9 activity kit active.@*Results@#(1) PQ could significantly reduced the survival rate of HK-2 cells and showed time and concentration dependence. The survival rate of HK-2 cells was about 55% after 800 μmol/L PQ contacted 24 h, XBJ under 20 mg/ml was no significant effect on the survival rate of HK-2 cells after cultured 72 h. (2) Compared with the PQ group, the survival rate of HK-2 cells of PQ+XBJ group was significantly increased (P<0.05). (3) Compared with the normal control group, the cell apoptosis rate of PQ group was significantly increased (P<0.05). Compared with the PQ group, the cell apoptosis rate of PQ+XBJ group was significantly decreased (P<0.05). (4) Compared with the normal control group, Bcl-2 protein expression in PQ group was significantly decreased and BAX protein expression in PQ group was significantly increased (P<0.05) ; compared with PQ group, Bcl-2 protein expression in PQ+XBJ group was significantly increased, BAX protein expression in PQ+XBJ group was significantly decreased (P<0.05). (5) Compared with the normal control group, the activities of caspase-3 and caspase-9 in PQ group were significantly increased (P<0.05). Compared with PQ group, the activities of caspase-3 and caspase-9 in PQ+XBJ group were decreased significantly (P<0.05) .@*Conclusion@#XBJ (10 mg/ml) has obvious protective effect on HK-2 cell injuried by PQ (800 μmol/L) , It can improve the survival rate of cells through reducing the apoptosis of HK-2 cells which induced by PQ.
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<p><b>OBJECTIVE</b>To investigate the protective effect of ulinastatin (UTI) on HK-2 cells during paraquat (PQ)-induced injury and its underlying mechanisms.</p><p><b>METHODS</b>Routinely cultured HK-2 cells were divided into blank control group, PQ group, UTI+PQ group and UTI group. Cell viability was determined by CCK-8 assay. The concentration of PQ in HK-2 cells were measured by high performance liquid chromatography (HPLC). The production of total reactive oxygen species (ROS) were detected by fluorescence microscopy. The activities of superoxide dismutase activity (SOD) and the content of malondialdehyde (MDA) in HK-2 cells were observed by chemical colorimetry. The levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>PQ, even at a dose of 200 µM, could significant suppress the viability of HK-2 cells in a dose-dependent and time-dependent. UTI showed no significant inhibitory effect on the viability of HK-2 cells when given at a dose below 8 000 U/ml (P > 0.05). Compared with the PQ group, the UTI+PQ group had significantly increased the viability of HK-2 cells in a dose-dependent of UTI (P < 0.05). Compared with the PQ group on the same hour, the UTI+PQ group showed decreased in PQ concentration in HK-2 cells (P < 0.05 for all except 6 h). Compared with the blank control group, the PQ group had significantly decreased SOD activity and significantly increased ROS level and MDA content (P < 0.05). Compared with the PQ group, the UTI+PQ group had significantly increased SOD activity and significantly decreased ROS level and MDA content (P < 0.05). Compared with the blank control group, the PQ group had significantly increased IL-6 and TNF-α level (P < 0.05); Compared with the PQ group, the UTI+PQ group had significantly decreased IL-6 and TNF-α level (P < 0.05).</p><p><b>CONCLUSION</b>UTI significantly reduces the PQ-induced oxidative damage and inflammatory injury and its mechanism may be by reducing the accumulation of PQ in HK-2 cells.</p>
Subject(s)
Humans , Cell Line , Cell Survival , Glycoproteins , Pharmacology , Interleukin-6 , Metabolism , Malondialdehyde , Metabolism , Oxidative Stress , Paraquat , Toxicity , Reactive Oxygen Species , Metabolism , Superoxide Dismutase , Metabolism , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of thalidomide in a mouse model of paraquat-induced acute lung injury and the mechanisms underlying the properties of thalidomide.</p><p><b>METHODS</b>Male ICR mice were randomly allocated into four groups: nomal control group (n = 30), thalidomide control group (n = 30), paraquat poisioning group (n = 30) and thalidomide treatment group (n = 90). Mice were sacrificed at 1d, 3d and 7d after paraquat poisioning. The level of (MDA) malondialdehyde, Superoxidedi-smutase (SOD) and glutathione (GSH) in the lung tissue were measuerd by chemical colorimetry. The expression of Nrf2 mRNA was determined by RT-PCR; Nuclear protein Nrf2 was abserved by Western blotting; Pathological changes of lung tissue were observed under light microscope by HE stain; the lung apoptosis cells were detected by TUNEL.</p><p><b>RESULTS</b>The levels of MDA, SOD and the expressions Nrf2 mRNA and protein Nrf2 in lung tissue were all markedly increased in mice of paraquat poisioning group than those in nomal group at 1 d, 3 d, 7 d. In contrast, the levels of GSH were decreaseel (P<0.05). Compared with paraquat poisioning group, the pulmonary SOD, Nrf2 mRNA and protein were increased and the lung wet dry ratio were all significantly decreased in mice of THD treatment group at 1 d, 3 d, 7 d (P<0.05). THD alleviated the pulmonary damage in the lightmicroscope at 3d after paraquat poisioning. The apoptosis index was markedly decreased in THD treatment groups comparing to paraquat piosioning group (P<0.05).</p><p><b>CONCLUSIONS</b>Lipid peroxide damage was one of the mechanisms of paraquat poisioning, thalidomide could attenuate paraquat-induced acute lung injury and its mechanism may be activating the Nrf2-ARE signaling pathway to protect mouse from Lipid peroxide damage.</p>