[Study on the Protective effect and mechanism of Nicotinamide Riboside on lung injury in paraquat intoxicated mice].

Objective: To investigate the protective effect and mechanism of Nicotinamide Riboside (NR) on lung injury caused by Paraquat intoxicated mice. Methods: Eighty clean male BALB/C mice were selected and averagely divided forty mice into 4 groups with 10 mice in each group, PQ group was given 25% PQ solution (60 mg/kg) by one-time gavage. PQ+NR group were intraperitoneally injected with NR solution (300 mg/kg) 1 hour before given the same amount of PQ solution (60 mg/kg) by one-time gavage, The Control group were given the same amount of saline by one-time gavage, The same amount of NR was intraperitoneally injected before NR group were given saline by one-time gavage. Observed and recorded general condition of PQ intoxicated mice. Observed and recorded the death of mice every half an hour and counted the mortality and drew survival curve of each group after 72 hours exposure. another forty mice were averagely divided and treated by the same way. After 24 hours of modelling, mice were anaesthetized and killed. Then blood was extracted after eyeball was removed. The changes of TNF-a、IL-6 and MPO in serum of mice were detected by ELISA.Two lung tissues were removed from the chest and used to measure the D/W ratio of the lung. The pathological changes of lung were observed and scored under light microscope.The levels of SOD, MDA and Caspase-3 in lung tissues were determined by chemical colorimetry. The expression of Sirt1 and Nrf2 in lung tissues was detected by Western-blot. Results: Compared with the Control group and the NR group, the mice in the PQ group had a poor general condition, such as depression, crouching, skin disorder and reduced activity, food, urine and feces. The symptoms in the PQ+NR group were reduced compared with the PQ group. The survival rate at 72 hours after exposure: 80% in the PQ+NR group and 40% higher than that in the PQ group (P=0.029) . Compared with Control group and NR group, the D/W ratio (0.09±0.07) , lung pathology score under light microscope (11.80±0.37) , TNF-a (39.89±1.48) pg/ml、IL-6 (77.29±2.38) pg/ml、MPO (0.31±0.01) µg/ml、SOD (6.62±0.30) U/mgprot、MDA level (1.21±0.14) mmol/mgprot, Caspase-3 activity (356.00± 27.16) %, Sirt1 and Nrf2 protein expression (1.02±0.14、0.82±0.06) were significantly decreased in PQ group (P=0.004、0.023) ; Compared with PQ group, PQ+NR group significantly increased the D/W ratio (0.10±0.10) , decreased the pulmonary pathology score under light microscope (7.400.51) , decreased TNF-a (33.00± 0.65) pg/ml、IL-6 (52.23±4.23) pg/ml、MPO leve (0.23±0.01) µg/mll, increased SOD leve (9.28±0.45) U/mgprotl, decreased MDA level (0.78±0.02) mmol/mgprot, decreased Caspase-3 activity (222.80±7.59) %, and increased the protein expressions of Sirt1 and Nrf2 (1.62±0.16、1.06±0.04) (P=0.048、0.035) . Conclusion: NR can prolong the survival time of PQ poisoned mice; NR intervention can effectively inhibit the inflammatory response, peroxidation injury and apoptosis of PQ poisoned mice; NR intervention can upregulate the expression of Sirt1 and Nrf2 protein and effectively reduce the lung injury of PQ poisoning.

[The protective effect of Xuebijing on paraquat-induced HK-2 cells apoptosis and the underlying mechanisms].

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.