Intervention Of Pyrrolidine Dithiocarbamate (PDTC) On The Paraquat-induced Pulmonary Injury In Rats

Paraquat (PQ) (1,10-dimethyl-4,40-bipyridinium dichloride), which is used world-widely for its high efficiency and low residues in the crops as a non-selective contact herbicide, can cause severe lung injury in humans and experimental animals. PQ-induced lung injury is characterized by initial destructive phase of pulmonary edema, alveolar hemorrhage, inflammatory infiltration, and second proliferative phase of fibroblast proliferation, excessive collagen deposition, which eventually results in pulmonary fibrosis and respiratory failure. So far, the mechanism of PQ toxicity is poorly understood and therapeutics has been disappointing and the mortality still remains high. Although the main agent of PQ toxicity has always been suggested to be reactive oxygen species (ROS), few studies have investigated the role of oxidative stress triggered-transcriptional factor regulation on cytokines in PQ-induced lung injury, which is most important to explore the treatment of PQ poisoning. This study investigate the dynamic changes of biomarkers of oxidative stress, transcriptional factors NF-kB and related cytokines, CTGF and its target genes over time, in order to explore the key points of PQ-induced pulmonary injury mechanisms in rat model. Moreover, we aimed to observe the effect of PDTC on PQ-induced lung injury and evaluate its potential therapeutic effect to provide scientific basis for the treatment strategy of PQ poisoning.We established acute PQ poisoning rat model by intragastric administration of 80mg/kg PQ.144 male Sprague-Dawley (SD) rats were randomly divided into four experimental groups:(1) Control group, n=6:animals treated with the saline solution; (2) PDTC group, n=36:animals administrated with saline solution.2h later injected with PDTC (100mg/kg); (3) PQ group, n=56:animals were intoxicated with aqueous solution of PQ (80mg/kg) by gastric gavage and followed injection of saline solution 2h later; (4) PQ+PDTC group, n=46:animals intoxicated with PQ (80mg/kg) and followed by injection of PDTC (100mg/kg) 2h later. Each group was sacrificed at the 1 st,3 rd,7 th,14 th,28 th, and 56 th day after treatment. The biomarkers of oxidative stress including malondialdehyde (MDA), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) in serum were measured using qualification kits. NF-?B p65-positive cells localization and NF-?B p65 expression intensity were detected by immunohistochemical tissue microarray. The nuclear factor-kappa B (NF-?B) activity in lung tissue was determined by electrophoretic mobility shift assay (EMSA). The levels of interleukin-1?(IL-1?), tumor necrosis factor-a (TNF-a), transforming growth factor-?1 (TGF-?1) and platelet-derived growth factor (PDGF) in serum were measured by ELISA. The protein expressions of connective tissue growth factor (CTGF) and a-smooth muscle actin (a-SMA) were determined by immunohistochemical SABC (streptavidin biotin peroxidase complex, SABC) and their dynamic changes were assayed by western blot. The mRNA levels of fibronectin (Fn), collagen I (ColI), and integrin a5 were analyzed with real-time quantitative PCR (RT-PCR). Meanwhile, the lung pathological changes were observed and semiquantified.The study on oxidative stress in acute PQ poisoned rats and effect of PDTC showed:The MPO activity, an index of neutrophils invasion and the lipid peroxidation marker MDA level were significantly increased and the activity of GPx, SOD, CAT in serum were significantly decreased in PQ group compared with control and PDTC group (P?0.05 or P?0.01) in the corresponding sacrifice dates. There were a significant decrease of MDA and increase of GPx, SOD, CAT in PQ+PDTC group compared with PQ group (P?0.05 or P?0.01) in the corresponding sacrifice dates.The study on NF-?B activity and dynamic changes of cytokines showed:(1) PQ induced a significant and time-dependent activation of NF-?B in rat lungs compared to control group. Tissue microarray showed NF-KB-positive cells were mainly expressed in cytoplasm or/and nucleus of bronchial mucosal columnar epithelial cells, the alveolar epithelial, macrophages, and neutrophils. EMSA showed the activity of NF-?B in lung tissue of PQ group significantly increased on the 1 st, the 3 rd, the 7 th, and 14 th day compared with control group (P?0.05 or P?0.01). PDTC treatment resulted in a significant reduction of PQ-induced NF-?B activation on the 1st, the 3 rd, and the 7 th day (P?0.01). (2) The level of IL-1?was significantly increased on the 1st, the 3 rd, and the 7 th day in PQ group compared with control and PDTC group (P?0.01). The levels of TGF-?1 and TNF-a in PQ group were significantly increased at all time points compared with control and PDTC group (P?0.05 or P?0.01). The level of PDGF significantly increased on the 7 th, the 14 th, the 28 th, and the 56 th day compared with control and PDTC group (P?0.01). Meanwhile, IL-1?and TNF-a were positively correlated with lung coefficient; TGF-?1, PDGF had positive correlation with hydroxyproline (Hyp). In the PQ+PDTC group, compared to PQ group, the levels of IL-1?, TGF-?1, TNF-a, and PDGF were significantly decreased in corresponding time points (P?0.05 or P?0.01).The study on expressions of CTGF and related profibrogenic genes indicated:(1) SABC immunohistochemistry showed CTGF positive cells mainly located in aleolar epithelial cells, endothelial cells, macrophages in early stage and fibroblasts were main positive cells on the 28 th and the 56 th day. a-SMA positive cells mainly located in fibroblasts. (2)The manner of CTGF, a-SMA expression was similar, detected by immunohistochemistry and western blot. With the time passing, their expressions in PQ group increased gradually compared with control group (P<0.05 or P?0.01). The increasing speed of CTGF, a-SMA was gentle on the 3 rd and the 7 th day. While their increasing speed were rapid from the 14 th to the 56 th day. PDTC treatment led to a significant decrease of CTGF and a-SMA compared with PQ group in corresponding time points (P?0.05 or P?0.01). (3) RT-PCR showed PQ significantly increased Fn mRNA level on all time points and increased intrgrin a5 mRNA level from the 3 rd to 56 th day compared with control group(P?0.05 or P?0.01). The ColI mRNA level significantly increased from the 3 rd to the 56 th day after PQ poisoning. PDTC treatment siganificantly deceased Fn, integrina5, and ColI mRNA levels (P?0.05 or P?0.01). (4) Correlation analysis showed CTGF was positively correlated with pathologic score or hydroxyproline (P?0.05 or P?0.01). CTGF was also positively correlated with a-SMA protein level, and Fn, integrin a5, Coll mRNA levels (P?0.01). a-SMA was positively correlated with pathologic score or hydroxyproline (P?0.01).The observation on PQ-induced lung structural and ultrastructural alterations showed:there were two distinct phases of pathological changes. (1) Observation at light microscopy (LM):destructive phase in early (1?7d) was characterized by hemorrhage, alveolar edema, and inflammatory cell infiltration. Proliferous phase in later stage (14?56d) was characterized by diffused alveolar collapse with an increased thickness of its wall, fibroblast proliferation and patchy distribution of collagen fibers. (2) Transmission electron microscopy (TEM) showed the swell, generation and necrosis of???alveolar epithelial cells; nucleic condensation and localization, swollen mitochondria, abundant rough endoplasmic reticulum (RER) and rich ribosome in the fibroblast. Noteworthy, in PQ+PDTC group, the occurrence of the above referred alterations were drastically attenuated.Hyp contents of the lung tissue in PQ group was significantly higher than control and PDTC group on the 14 th, the 28 th and the 56 th day(P?0.01), and its content showed lower in PQ+PDTC group on the 28 th and the 56 th day compared with PQ group(P<0.01). PQ induced gradually increased lung coefficient over time. It had a rapid decrease on the 14 th day but showed a increasing trend on the 28 th and 56 th day again. The lung coefficient in PQ group was significantily higher than control group on all time points (P<0.01).There was a significant decreased coefficient in PQ+PDTC group on the 3 rd,7 th,28 th, and 56 th day compared with PQ group (P?0.01).In conclusion, it suggested that oxidative stress plays important role in PQ-induced acute lung injury at early stage. As one NF-?B stimulates, it futher activates NF-?B and thus causes aberrant regulation of many cytokines such as IL-1(3, TNF-a, TGF-?1, PDGF involved in development of PQ-induced ALI/ARDS and fibrosis. Among them, TGF-?1 acts as a "key player" of pulmonary fibrosis. Its overexpression may eventully induces CTGF expression through multiple sigaling pathways. CTGF plays the role as a downstream regulator of TGF-?1 to induce lung fibrosis, which is characterized by increased a-SMA protein level, and Fn, ColI, integrin a5 mRNA levels. PDTC, as a strong antioxidant and NF-?B inhibitor, it dramastically corrects PQ-induced imbalance of oxidation/antioxidation as well as inhibits NF-?B activity. Therefore, it significantly decreased expressions of some cytokines and fibrogenic genes, leading to drastically attenuated pulmonary inflammation and fibrosis. So, intervention of PDTC not only proved our hypothesis of PQ poisoning mechanism but also provided clues for therapy. However, the mechanisms of PDTC intervention still remain to be explored.