百草枯中毒致肺纤维化大鼠细胞因子表达及四氢吡咯二硫代氨基甲酸酯的干预作用

Changes of Fibrogenic Cytokines in Acute Paraquat Poisoned Rats and Effects of Pyrrolidine Dithiocarbamate

目的探讨百草枯中毒致肺纤维化的机制。方法 SD大鼠随机分为对照组6只、四氢吡咯二硫代氨基甲酸酯(PDTC)对照组36只、百草枯染毒组36只、PDTC干预组36只。染毒组和PDTC干预组给予百草枯80 mg/kg一次性灌胃后2 h,染毒组给予等量生理盐水腹腔注射,PDTC干预组给予PDTC 100 mg/kg一次性腹腔注射;对照组和PDTC对照组予生理盐水1 mL/kg灌胃后2 h,对照组给予等量生理盐水腹腔注射,PDTC对照组给予PDTC 100 mg/kg一次性腹腔注射。于不同处理后1、3、7、14、28、56 d观察大鼠中毒表现,并分别处死6只大鼠观察肺组织病理改变,测定肺组织羟脯氨酸含量,测定血清胰岛素样生长因子1(IGF-1)、转化生长因子β1(TGF-β1)、血小板源性生长因子(PDGF)水平及肺组织结缔组织生长因子(CTGF)的表达,分析它们与肺组织羟脯氨酸含量的关系。结果染毒早期(1～7 d)病理特征为急性肺泡炎,表现为肺充血、肺水肿及炎性细胞浸润。后期(14～56 d)炎性病变减轻,支气管周围、肺泡间隔及肺泡内大量成纤维细胞增殖、胶原纤维增生。干预组的肺部炎性改变及胶原纤维增生程度均明显减轻。与对照组比较,染毒组IGF-1含量3～56 d明显升高(P<0.01),TGF-β1含量各时段均明显升高(P<0.01),PDGF含量7～56 d显著升高(P<0.01)。经PDTC干预后,上述细胞因子水平明显降低,相应时点差异有统计学意义(P<0.05或P<0.01)。免疫组化显示,染毒组3 d即有CTGF阳性表达,表达强度较弱,主要表达于炎症细胞;7、14 d表达进一步增强,28、56 d表达持续增强,主要表达于巨噬细胞和成纤维细胞。干预组CTGF阳性表达细胞与染毒组相同,但各时段表达强度均显著降低(P<0.05或P<0.01)。结论细胞因子IGF-1、TGF-β1、PDGF及CTGF的过度表达是参与百草枯致肺纤维化的重要机制。PDTC可能通过抑制NF-κB活化进而抑制上述细胞因子的表达,减轻百草枯中毒大鼠的肺损伤和肺纤维化。

Objective To investigate the mechanism of lung injury caused by paraquat poisoning by observing the changes of fibrogenic cytokines in acute paraquat poisoned rats and the effects of pyrrolidine dithiocarbamate （PDTC）. Methods Sprague-Dawley rats were randomly divided into three groups, ie. a control group （ n = 6）, a PDTC group （ n = 36 ）, a paraquat group （ n = 36 ）, and a paraquat ＋ PDTC group （n = 36）. The rats in the PDTC group,the paraquat group, and the paraquat ＋ PDTC group were subdivided into 6 subgroups sacrificed respectively on 1 st ,3rd ,7th, 14,h ,28,h and 56th day after the treatment. The levels of transforming growth factor-β1 （ TGF-β1 ）, platelet-derived growth factor （ PDGF）, insulin-like growth factor-1 （IGF-1） in serum were measured. Meanwhile the expression of connective tissue growth factor （CTGF） and hydroxyproline in lung tissues were detected. The relationship of above cytokines with hydroxyproline was analyzed. Results The destructive phase in early （1-7 d） was characterized byhemorrhage, alveolar edema, and inflammatory cell infiltration. The proliferous phase in later stage （ 14 56 d） was characterized by diffused alveolar collapse with fibroblast proliferation and patchy distribution of collagen fibers. Compared with the control group,the level of TGF-β1 on all time points,the level of PDGF from 7th to 56th day,the level of IGF-1 from 3rd to 56th day in the paraquat group all significantly increased （P 〈 0. 01 ）. Immunohistochemistry results showed CTGF positive cells mainly located in aleolar epithelial ceils, endothelial cells, macrophages in early stage, and fibroblasts were main positive cells on the 28th and the 56th day. The expression of CTGF in the paraqnat group increased gradually compared with the control group on different time points （P 〈 0. 05 or P 〈 0. 01 ）. Meanwhile, the levels of above cytokines were positively correlated with the level of hydroxyproline. Noteworthy, PDTC treatment led to significant decreases of above cytokines compared with the paraquat group in corresponding time points （ P 〈 0. 05 or P 〈 O. O1 ）. Conclusions Over expressions of IGF-1, TGF-β1, PDGF, IGF-1 and CTGF may play important roles in lung fbrosis of paraquat poisoned rats. PDTC, as a strong NF-KB inhibitor, may inhibits NF-KB activity and further significantly decreases expressions of cytokines, leading to significantly attenuated pulmonary inflammation and fibrosis. However,the mechanisms of PDTC intervention still remain to be explored.