WIN55212-2对百草枯中毒小鼠肺损伤的保护作用

摘要

Objective To explore the protective effects of cannabinoid analogues WIN55212-2 on paraquat poisoned mice. Methods Totally 35 healthy male C57BL/6 mice were randomly(random number) divided into four groups: PQ group (paraquat poisoned, n=10), WIN 1 mg group (PQ+WIN55212-21 mg n=10), WIN 2 mg group (PQ+WIN55212-22 mg, n=10), control group (n=5).The PQ poisoned animal models were established in the PQ group, WIN 1 mg group and WIN 2 mg group by intraperitoneally injection of paraquat with a concentration of 20 mg/kg. Intraperitoneal injection of WIN55212-2 (containing Tween 80 cosolvent) at the concentration of 1 mg/kg and 2 mg/kg was performed 1 h before PQ exposure in the two interfered groups. Equivalent volume of saline was given to the control group. WIN55212-2 was injected twice a week from the second week. In the acute phase (14 d), 5 mice were randomly sacrificed in the PQ group, WIN 1 mg group and WIN 2 mg group, and 3 mice were sacrificed in the control group to obtain blood sample, bronchoalveolar lavage fluid (BALF) and lung tissue. All the remaining mice were executed on day 28, and the tissue samples were collected as mentioned above. HE staining and Masson staining were performed to observe the changes of lung tissues after PQ poisoning. Changes of TNF-α, IL-6 and TGF-β in plasma and BALF were measured by ELISA. Results In the acute phase, the pathological sections of lung tissues in the PQ group, WIN 1 mg group and WIN 2 mg group showed diffuse inflammation, which was improved after the intervention of WIN5522-2, especially in the WIN 1 mg group. IL-6 levels of BALF in the PQ group, WIN 1 mg group, WIN 2 mg group and the control group were (1024.77±124.74)U/L, (620.48±99.76)U/L, (823.29±157.88) U/L, and (180.42±20.22)U/L, respectively. IL-6 levels in the WIN 1 mg group and the WIN 2 mg group were statistically lower than those in the PQ group (P=0.021, P=0.016). However, no difference was found between the two intervention groups(P=0.114). The similar condition was also found in TNF-α in BALF and plasma. In the chronic phase, mice in the PQ group, WIN 1 mg group and WIN 2 mg group showed fibrosis in tissue by HE and Masson staining, and the inflammatory condition was improved after the intervention of WIN5522-2, which was more obvious in the WIN 1 mg group. In BALF, TNF-α level was (321.64±50.54)U/L, (260.23±48.19)U/L, (278.89±29.40)U/L, (89.76 ± 10.87)U/L in the PQ group, WIN 1 mg group, WIN 2 mg group and the control group. Differences were found between the WIN 1 mg group and the control group and the WIN 2 mg group. Similar differences were also observed in plasma TNF-α, but not in TGF-β. Conclusions A small dose of WIN55212-2 can improve the general condition of PQ poisoning mice, and reduce the inflammatory and fibrosis-related cytokines levels in PQ poisoning mice.%目的 探讨大麻素受体激动剂WIN55212-2对百草枯(paraquat,PQ)中毒模型小鼠肺损伤的保护作用.方法 健康成年雄性C57BL/6小鼠35只,随机(随机数字法)分为4组:百草枯中毒组(PQ组,n=10),PQ+WIN55212-21 mg干预组(WIN 1 mg组,n=10),PQ+WIN55212-22 mg干预组(WIN 2 mg组,n=10),对照组(control组,n=5).PQ组、WIN 1 mg组、WIN 2 mg组建立百草枯中毒模型,于实验第1天和第3天腹腔注射PQ 20 mg/kg.WIN 1 mg组和WIN 2 mg组分别于PQ染毒前1 h按1 mg/kg和2 mg/kg的浓度经腹腔注射WIN55212-2(含Tween 80助溶剂),PQ组和control组注射等容量的生理盐水和Tween 80液.从第2周开始,干预药物每周注射两次.急性期(14 d)随机处死PQ组、WIN 1 mg组、WIN 2 mg组各5只小鼠,对照组处死3只,取血标本,离心得血浆,取肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和肺组织.剩余存活小鼠均在28 d时处死,再次留取上述组织标本.肺组织行HE染色、Masson染色,观察PQ中毒后小鼠的肺组织变化.酶联免疫吸附法(ELISA)测定急性期小鼠血浆和BALF中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)及慢性期血浆和BALF中TNF-α 和转化生长因子 β(TGF-β)的含量变化.结果 急性期:PQ组、WIN 1 mg组、WIN 2 mg组小鼠肺组织病理切片均呈现弥漫性炎症,予WIN55212-2干预后炎症有所改善,尤以WIN 1 mg组更为明显.PQ组、WIN 1 mg组、WIN 2 mg组,对照组BALF中IL-6水平(U/L)分别为1024.77±124.74、620.48±99.76、823.29±157.88、180.42±20.22;WIN 1 mg组和WIN 2 mg组均较PQ组下降,且差异有统计学意义(P=0.021,P=0.016),但两干预组间差异无统计学意义(P=0.114).急性期BALF和血浆中TNF-α 水平与上述情况相似.慢性期:PQ组、WIN 1 mg组、WIN 2 mg组小鼠肺组织HE及Masson染色可见明显纤维化,予WIN55212-2干预后可见炎症情况有所改善,以WIN 1 mg组更为明显.慢性期PQ组、WIN 1 mg组、WIN 2 mg组小鼠BALF中TNF-α 水平(U/L)分别为321.64±50.54、260.23±48.19、278.89±29.40、89.76±10.87,WIN 1 mg组与对照组及WIN 2 mg组间差异有统计学意义.相似的情况可见于慢性期血浆TNF-α,但TGF-β 未见这种差异.结论 小剂量WIN55212-2可以改善PQ中毒小鼠的一般状况,降低PQ中毒小鼠炎症和纤维化相关细胞因子水平,WIN55212-2可能对百草枯中毒模型小鼠肺损伤具有保护作用.