烟酰胺核糖对百草枯中毒小鼠肺损伤的保护作用及机制研究

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

目的探讨烟酰胺核糖(Nicotinamide riboside,NR)对百草枯(Paraquat,PQ)中毒小鼠肺损伤的保护作用及其机制。方法于2017年4至7月,选择清洁级雄性BALB/C小鼠80只,将其中40只小鼠随机均分4组,每组10只。PQ组:以25%PQ溶液(60 mg/kg)灌胃染毒;PQ+NR组:等量灌胃染毒,且灌胃前1 h腹腔注射(300 mg/kg)的NR;Control组:等量生理盐水灌胃;NR组:等量生理盐水灌胃,且灌胃前腹腔注射等量的NR(300 mg/kg)。观察并记录PQ中毒小鼠一般状况,同时每半小时观察并记录,统计染毒后72 h的各组死亡数,计算各组小鼠生存曲线。将另40只小鼠同样方法进行造模和分组,PQ灌胃后24 h,麻醉必死,摘眼球留取血标本用酶联免疫吸附(ELISA)等方法检测小鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、髓过氧化物酶(MPO)的变化。开胸取肺组织,测肺干湿重比(D/W)比值,光镜下观察肺病理变化并评分,用化学比色法检测肺组织超氧化物歧化酶(SOD)、丙二醛(MDA)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)水平,免疫印迹法(Western-blot)检测肺组织沉默调节蛋白1抗原(Sirt1)和核因子E2相关因子2(Nrf2)表达变化情况。结果Control组和NR组小鼠的一般状况和行为正常;与Control组和NR组比较,PQ组小鼠一般状况差,精神萎靡、蜷缩、皮毛紊乱,活动、进食、大小便减少;与PQ组比较,PQ+NR组小鼠各症状均减轻。染毒后72 h的存活率:与PQ组(40%)比较,PQ+NR组(80%)存活率提高(P=0.029)。与Control组和NR组比较,PQ组小鼠D/W比值明显下降、光镜下肺病理学评分明显上升,TNF-α、IL-6、MPO水平明显上升,SOD水平明显下降,MDA水平明显上升;Caspase-3活力明显上升(P<0.01)。Sirt1和Nrf2蛋白表达量(1.02±0.14、0.82±0.06)明显下降(P=0.004、0.023)。与PQ组比较,PQ+NR组D/W比值(0.10±0.10)明显上升,光镜下肺病理学评分(7.40±0.51)下降,TNF-α(33.00±0.65)pg/ml、IL-6(52.23±4.23)pg/ml、MPO(0.23±0.01)μg/ml水平明显下降,SOD(9.28±0.45)U/mgprot水平明显升高,MDA(0.78±0.02)mmol/mgprot水平明显下降,Caspase-3(222.80±7.59)%活力明显降低,Sirt1和Nrf2蛋白表达量(1.62±0.16、1.06±0.04)明显升高(P=0.048、0.035)。结论PQ中毒可致小鼠急性肺损伤,而NR能抑制PQ中毒小鼠炎症反应、氧化损伤及细胞凋亡,对PQ中毒所致的肺损伤具有一定的保护作用,可能是通过上调Sirt1、Nrf2蛋白的表达来实现的。

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.