白藜芦醇对百草枯中毒小鼠肺损伤的干预及机制

The effect of resveratrol on paraquat-induced acute lung injury in mice and its mechanism

目的观察用白藜芦醇（Res）干预对百草枯（PQ）中毒小鼠死亡率和肺损伤的影响，探讨核转录因子-κB（NF—κB）炎症通路在其中的作用机制。方法68只SPF级健康雄性ICR小鼠，20只用于观察死亡情况（每组10只），48只用于指标检测（每组6只）。按随机数字表法将小鼠分为生理盐水（NS）对照组、Res对照组、PQ组和PQ＋Res组，后两组再分为6、24、72h亚组。一次腹腔注射30mg／kgPQ建立PQ中毒小鼠模型；于小鼠染毒后立即于对侧腹腔注射Res60mg／kg进行干预。分别于染毒后6、24、72h取血后活杀小鼠留取肺组织，用酶联免疫吸附试验（ELISA）检测血清肿瘤坏死因子-α（TNF—α）、白细胞介素（IL-6、IL-1β）含量；电镜下观察肺组织结构变化；原位末端缺刻标记试验（TUNEL）检测细胞凋亡，并计算细胞凋亡率；蛋白质免疫印迹试验（Western Blot）检测胞核内NF—κBp65蛋白表达。结果与PQ组相比，PQ＋Res组小鼠48、72、96h累计死亡数有所下降（分别为0比2、2比5、4比6），但差异无统计学意义（均P〉0．05）.电镜下观察可见，PQ组肺组织损伤较NS对照组明显加重，Res干预能够减轻小鼠肺组织损伤程度。与NS对照组[（2．45±0．61）％]相比，PQ组6h肺组织细胞凋亡率即明显增加（（8．42±1．48）％]，72h达峰值[（21．23±3．47）％]；Res干预能够有效减低PQ中毒后小鼠肺组织细胞的凋亡率[6h：（5．56±1．31）％比（8．42±1．48）％，24h：（11．14±2．07）％比（16．88±2．96）％，72h：（13．28±2．32）％比（21．23±3．47）％，均P〈0．05]。PQ中毒后血清TNF—α、IL-6和IL-1β水平以及肺组织细胞核NF—κBp65表达均明显增加；Res干预后上述各指标均较PQ组明显降低[TNF—α．（ng／L）：6h为2．62±0．29比4．06±0．74，24h为3．98±0．41比6．79±0．80，72h为5．06±0．75比11．00±0．75；IL-6（ng／L）：6h为14．19±1．54比16．55±1．24，24h为13．21±1.37比19．73±0．85，72h为13．72±0．56比22．45±0．72；IL-1β（ng／L）：6h为8．54±1．64比12．59±0．66，24h为10．15±0．29比16．24±1．03，72h为16．14±0．70比19．55±0．56；6hNF—κBp65（以NS对照组为1）：（1．34±0．07）倍比（1．86±0．11）倍；均P〈0．05]。结论Res干预不能避免PQ中毒小鼠的死亡，但能够减轻肺组织损伤程度和细胞凋亡。Res对PQ中毒肺损伤的保护机制与其对NF—κB065炎症反应通路的抑制效应有关。

Objective To investigate the effect of resveratrol （Res） on paraquat （PQ）-induced acute lung injury （ALI） and mortality in mice and the mechanism of nuclear factor-κB （NF-κB） inflammatory pathway. Methods Sixtyeight healthy male ICR mice with grade SPF were enrolled, among them 20 mice were used for mortality observation （n = 10）, and other 48 were used for determination of related parameters （n = 6）. The mice were randomly divided into four groups： normal saline （NS） control group, Res control group, PQ group and PQ ＋ Res group. The mice in the latter two groups were subdivided into 6, 24, 72 hours subgroups. The PQ poisoning model of mice was reproduced by one injection of 30 mg/kg PQ intraperitoneally. The mice in PQ ＋ Res group were given 60 mg/kg Res intraperitoneally on the contralateral side after PQ injection. The mice were sacrificed at 6, 24, 72 hours after PQ poisoning, and lung tissue was harvested. The serum levels of tumor necrosis factor- α （TNF- α ）, interleukins （IL-6 and IL-1β ） were determined by enzyme linked immunosorbent assay （ELISA）. The pathological changes in lung tissue were observed with electron microscopy. Apoptosis cells in the lung were identified by terminal dexynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） for the estimation of apoptosis rate. The protein expression of NF-KB p65 was determined by Western Blot. Results Compared with PQ group, the death number of mice at 48, 72, 96 hours in PQ ＋ Res group was slightly deereased （0 vs. 2, 2 vs. 5, 4 vs. 6） but without statistically significant difference （all P 〉 0.05）. Under eleetron mieroseope, the lung injury in PQ group was severer than that in NS control group, and Res was found to be able to alleviate the lung injury. Compared with NS control group [（2.45 ± 0.61）%], the apoptosis rate at 6 hours in PQ group was signifieantly inereased [（8.42±1.48）%], and peaked at 72 hours [（21.23 ±3.47）%]. Res could deerease the apoptosis rate after PQ poisoning [6 hours： （5.56± 1.31）% vs. （8.42±1.48）%, 24 hours： （11.14± 2.07）% vs. （16.88±2.96）%, 72 hours： （13.28± 2.32）% vs. （21.23± 3.47）%, all P 〈 0.05]. The serum levels of TNF- α, IL-6, and IL-1 β, and NF-κB p65 in lung tissue were all markedly increased after PQ poisoning, and they were significantly decreased after Res intervention as compared with those of PQ group [TNF- α （ng/L）： 2.62±0.29 vs. 4.06±0.74 at 6 hours, 3.98±0.41 vs. 6.79±0.80 at 24 hours, 5.06±0.75 vs. 11.00±0.75 at 72 hours; IL-6 （ng/L）： 14.19±1.54 vs. 16.55 ±1.24 at 6 hours, 13.21±1.37 vs. 19.73 ±0.85 at 24 hours, 13.72±0.56 vs. 22.45±0.72 at 72 hours; IL-1 β（ng/L）： 8.54±1.64 vs. 12.59±0.66 at 6 hours, 10.15±0.29 vs. 16.24±1.03 at 24 hours, 16.14 ±0.70 vs. 19.55 ±0.56 at 72 hours; 6-hour NF-κB p65： （1.34± 0.07） folds vs. （1.86±0.11） folds when the expression in NS control group was represented as 1, all P 〈 0.05]. Conclusions Res eannot lower the mortality in mice with PQ poisoning, but it seems to be able to attenuate PQ-indueed ALI and eel1 apoptosis. The mechanism responsible for the latter maybe the inhibitive effect of Res on NF-κB p65 transloeation and eytokines production.