吡啡尼酮对百草枯中毒致急性肺损伤小鼠的抗氧化作用

Anti-oxidant effect of pirfenidone on acute lung injury induced by paraquat poisoning in mice

目的：探讨不同剂量吡啡尼酮（PD）对百草枯（PQ）中毒致急性肺损伤（ALI）小鼠的抗氧化作用。方法将144只ICR小鼠按随机数字表法分为对照组（n＝24）、PQ中毒组（n＝24）及高、低剂量PD干预组（n＝48）。采用一次性腹腔注射20%PQ溶液25mg/kg制备PQ中毒致ALI模型；对照组给予等量生理盐水。低、高剂量PD干预组于染毒后即刻分别灌胃PD混悬液30mg/kg、70mg/kg〔PD溶于0.4%羧甲基纤维素钠（CMC）溶液中〕，连续3d；对照组和PQ中毒组灌胃等量0.4%CMC溶液。各组分别于灌胃后2、6、12、24、48、72h处死小鼠取肺组织，采用酶联免疫吸附试验（ELISA）检测核转录因子-κB（NF-κB）水平；采用比色法检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量；苏木素-伊红（HE）染色后光镜下观察肺组织病理学改变。结果与对照组比较，PQ中毒组2h起NF-κB即显著升高（pg/mg：106.65±5.96比79.04±2.40，P＜0.05），并持续至72h（pg/mg：110.47±5.91比82.70±2.79，P＜0.05）；SOD活性在早期（2～6h）即明显增高（U/mg：2h为39.34±1.17比34.72±1.54，6h为37.37±0.90比33.75±0.93，均P＜0.05），然后逐渐下降；MDA于24h内显著升高（nmol/mg：2h为1.67±0.22比1.03±0.09，24h为1.56±0.17比1.14±0.16，均P＜0.05），随后逐渐下降。与PQ中毒组比较，高、低剂量PD干预组在24～72h能显著抑制NF-κB升高，24h内能显著抑制MDA含量升高；高剂量PD干预组能提高6hSOD活性，而低剂量PD干预组SOD活性呈先降低后升高趋势。与高剂量PD干预组比较，低剂量PD干预组MDA含量进一步降低（nmol/mg：2h为0.90±0.08比1.29±0.18，6h为1.03±0.32比1.84±0.43，24h为1.08±0.09比1.33±0.16，均P＜0.05）；SOD活性在6h显著降低（U/mg：35.24±2.08比38.46±0.87，P＜0.05），72h有所回升（U/mg：39.81±1.30比34.58±3.15，但P＞0.05）；而各时间点NF-κB水平差异无统计学意义。光镜下观察，PQ中毒组早期即有大量红细胞、炎性细胞浸润及浆液渗出，并可见部分肺泡破裂；高、低剂量PD干预组病理学改变较PQ中毒组明显减轻，但不同剂量组间无明显差异。结论 PD可抑制PQ中毒大鼠早期肺部炎症的发展，其机制可能与抑制活性氧及NF-κB活化有关；低剂量PD较高剂量PD的治疗效果可能更好。

Objective To investigate the anti-oxidant effect of pirfenidone （PD） at different dosage on acute lung injury （ALI） induced by paraquat （PQ） poisoning in mice. Methods 144 ICR mice were randomly divided into four groups： control group （n = 24）, PQ poisoned group （n = 24）, high and low doses PD treatment groups （n = 48）. ALI induced by PQ poisoning model was reproduced by intraperitoneal injection of 25 mg/kg 20% PQ solution in mice, and the mice in control group was given equal volume of normal saline. Intragastric administration with 30 mg/kg and 70 mg/kg PD suspension [PD was dissolved in 0.4% sodium carboxymethyl cellulose sodium （CMC） solution] after PQ poisoning immediately for 3 days in high and low doses PD treatment groups respectively, while the same volume of 0.4% CMC solution was administrated in control group and PQ poisoned group. Then mice in each group were respectively sacrificed at 2, 6, 12, 24, 48 and 72 hours after PD exposure to harvest the lung tissue, nuclear factor-κB （NF-κB） was determined by enzyme linked immunosorbent assay （ELISA）, superoxide dismutase （SOD） and malonaldehyde （MDA） were determined by colorimetry, and pulmonary pathological changes were observed with microscope after hematoxylin-ensin （HE） staining. Results Compared with the control group, NF-κB from 2 hours in PQ poisoned group was significantly increased （pg/mg： 106.65±5.96 vs. 79.04±2.40, P 〈 0.05）, and lasted to 72 hours （pg/mg： 110.47±5.91 vs. 82.70±2.79, P 〈 0.05）; the activity of SOD was significantly enhanced in early stage （2-6 hours; U/mg： 39.34±1.17 vs. 34.72±1.54 at 2 hours, 37.37±0.90 vs. 33.75±0.93 at 6 hours, both P 〈 0.05） followed by a gradual decrease; the content of MDA within 24 hours was significantly increased （nmol/mg： 1.67±0.22 vs. 1.03±0.09 at 2 hours, 1.56±0.17 vs. 1.14±0.16 at 24 hours, both P 〈 0.05） followed by a gradual decrease. Compared with the PQ poisoned group, both high and low dose PD treatment could significantly inhibit NF-κB from 24 hours to 72 hours, and significantly inhibit MDA within 24 hours; high dose PD treatment could increase SOD activity at 6 hours, which showed a tendency of decreasing followed by increasing in low dose PD treatment group. Compared with high dose PD treatment group, the inhibition of MDA in low dose PD treatment group was more significant （nmol/mg： 0.90±0.08 vs. 1.29±0.18 at 2 hours, 1.03±0.32 vs. 1.84±0.43 at 6 hours, 1.08±0.09 vs. 1.33±0.16 at 24 hours, all P 〈 0.05）, and SOD activity was significantly decreased at 6 hours （U/mg： 35.24±2.08 vs. 38.46±0.87, P 〈 0.05）, and it was increased at 72 hours （U/mg： 39.81±1.30 vs. 34.58±3.15, but P 〉 0.05）, but no significant difference in NF-κB activity at all time points was found. Under light microscope, a wide range of red blood cells and serous effusion, alveolar septum fracture and pulmonary interstitial inflammatory cell infiltration were shown by pathologic examination in PQ poisoned group. The pathologic changes in high and low doses PD treatment groups were obviously less than those of PQ poisoned group, and no significant difference was found between the two doses groups. Conclusions The early therapeutic effect of PD may relate to the inhibition of NF-κB and reactive oxygen species, then reduce the inflammation of PQ poisoning. The treatment effectiveness of low dose PD seems better than high dose PD.