Pyrrolidine dithiocarbamate attenuates paraquat-induced acute pulmonary poisoning in vivo via transforming growth factor β1 and nuclear factor κB pathway interaction

Abstract

Paraquat (PQ) exposure could cause pulmonary fibrosis. The aim of this study was to investigate the protective effect of pyrrolidine dithiocarbamate (PDTC) in an acute PQ poison model. One hundred and forty-four Sprague Dawley rats were equally divided into three experimental groups: control group, PQ group, and PQ + PDTC group. At days 1, 3, 7, 14, 28, and 56 of treatment, the serum levels of transforming growth factor β1 (TGF-β1), the levels of hydroxyproline, the protein expression of nuclear factor κB (NF-κB) pathway, and histopathological change in lung tissue were assessed. The survival rate of rats treated with PQ + PDTC was increased compared with that of rats treated only with PQ (p < 0.05), and the occurrence of pathological changes was dramatically attenuated in the PQ + PDTC group. The serum levels of TGF-β1 and the hydroxyproline levels in the PQ group were significantly increased in a time-dependent manner compared with those in the control and PQ + PDTC groups on days 7, 14, 28, and 56 (p < 0.05). Additionally, the protein levels of NF-κB proteins p65, inhibitor of κB (IκB) kinase (IKKβ, and IκB-α were significantly downregulated in the PQ + PDTC group as determined by array analysis. The present findings suggest that overexpression of TGF-β1 may play an important role in PQ-induced lung injury and that PDTC, a strong NF-κB inhibitor, can rescue PQ-induced pulmonary fibrosis by influencing the protein expression of NF-κB pathway.

Introduction

Paraquat (PQ) is a popular herbicide worldwide and has been used for centuries. However, the serious pulmonary fibrosis caused by long-term accumulation of the chemical through the skin, ingestion, or inhalation makes it one of the most dangerous toxicants and a detriment to human health. ¹ Although the exact mechanism of toxicity is unclear, oxidative stress and lipid peroxidation are believed to comprise the initial step. PQ-induced lung injury is characterized by pulmonary fibrosis and subsequent respiratory failure. ^2,3 This lung tissue damage is achieved by inflammatory cell stimulation, fibroblast proliferation, and excessive collagen deposition. ⁴ Unfortunately, nowadays no specific therapy agent can absolutely treat PQ poisoning, and this explains the high prevalence of PQ-induced mortality. ⁵ Alternatively, an optimal treatment strategy for PQ-induced lung injury is to suppress production of reactive oxygen species (ROS) and regulate fibrosis. ⁶

Pyrrolidine dithiocarbamate (PDTC) is a chemical compound that is used for diverse biochemical applications including cell cycle regulation, heavy metal chelation, and enzyme inhibition. ^7,8 Traditionally, PDTC has been used as an antioxidant compound to enhance the oxidative stress and antioxidant defense systems during activation of endogenous antioxidant gene expression, including genes for superoxide dismutase ⁹ and γ-glutamylcysteine synthetase. ¹⁰ In contrast, PDTC may inhibit malondialdehyde formation, a marker for lipid peroxidation. ¹¹ Furthermore, PDTC has also been documented to serve as a nuclear factor κB (NF-κB) inhibitor ^12,13 and can interfere with several pro-inflammatory cytokines through the NF-κB pathway. ¹⁴ NF-κB is a transcription factor ubiquitously expressed in mammalian cells, which regulates the inflammatory response.

Considering the characteristic of counteracting the toxic effects of free radicals and immunomodulatory effects on NF-κβ activation, it may be beneficial for treating acute lung injury caused by PQ exposure. Accordingly, the current study aimed to evaluate the antagonistic effects of PDTC on lung fibrosis induced by PQ and further investigate the interactive roles of transforming growth factor β1 (TGF-β1) and the NF-κB pathway in this progression (supplementary figure).

Materials and methods

Chemicals

PQ (45%, methyl viologen) was provided by Sinon Chemical (Shanghai, China). PDTC and all other chemical reagents were purchased from Sigma-Aldrich (St Louis, Missouri, USA).

Animal model and study group

One hundred and forty-four male Sprague Dawley rats (180–220 g each) were purchased from the Laboratory Animal Research Center of Fudan University. The animals were housed in stainless steel cages under a 12-h light/12-h dark cycle. The environment was climate-controlled for temperature and humidity, and they were allowed access to a pellet diet and tap water ad libitum for the duration of the study. Animals were equally divided into a control group (animals received saline only), a PQ group (animals were orally given an aqueous solution of PQ at 40  mg/kg), and a PQ + PDTC group (animals were subjected to 40  mg/kg PQ, followed by an immediate intraperitoneal (IP) injection of PDTC at 120  mg/kg). These experimental groups consisted of 48 animals each. About 10 animals from each group were then killed at days 3, 7, 14, 28, and 56. All the procedures in this study were performed in accordance with the guidelines of the Helsinki Convention for the use and care of animals as adopted by the Ethics Committee of Fudan University.

Tissue preparation

During the study, a total of 6  mL of venous blood was collected in an ethylenediaminetetraacetic acid K3-containing Vacutainer on days 3, 7, 14, 28, and 56; rats were then killed with an IP injection of 10% chloralum hydratum (3  mL/kg). The left lung was fixed in a distended state by infusion of 10% neutrally buffered formalin into the trachea and incubation in an adequate amount of 10% neutrally buffered formalin for 1 week. The lung tissues were processed by an automatic tissue processor, embedded in paraffin blocks, cut into 4 μm slices, and stained with hematoxylin and eosin (H&E) and Masson’s trichrome for collagen. For the histological examinations, all slides were assessed by a pathologist blinded to the experimental parameters. Four pathological features, including alveolar capillary congestion, alveolar hemorrhage, infiltration, and accumulation of neutrophils in the airspace or vessel wall and hyaline membrane formation with thickening of the alveolar wall, were graded on a 5-point scale (0–4) for damage as described elsewhere by Henes et al ¹⁵ : 0 (minimal), 1 (mild), 2 (moderate), 3 (severe), and 4 (maximal). A total lung injury score was calculated as the sum of the scores of all four pathological features.

Hydroxyproline levels in lung tissue and TGF-β1 levels in plasma

The hydroxyproline levels in the lung tissues of control, PQ, and PQ + PDTC group rats were examined using commercial kits (Sigma), and the data are reported as nanogram per gram wet lung tissue. Serum TGF-β1 levels were detected using a commercial enzyme-linked immunosorbent assay kit (R&D, Minneapolis, MN, USA) per manufacturer’s instructions.

Protein array

The NF-κB pathway antibody microarray contains 1318 antibodies along with positive and negative controls, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, California, USA) according to their established protocol. ¹⁶ The procedure was performed as described previously. ¹⁷ Briefly, the slides were first blocked in a blocking solution for 30 min at room temperature, washed with phosphate-buffered saline for 5 min and air-dried. Array slides were then incubated with biotin-labeled cell lysates in a binding solution for 2 h at room temperature and washed 3 times with wash solution. The GenePix 4000 scanner was used to scan Cy3-conjugated antibody signals, and data were analyzed by GenePix Pro 6.0 (Molecular Devices, Sunnyvale, California, USA). The fluorescence intensity of each antibody spot was normalized to the blank signal.

Statistical analysis

SPSS 13.0 (SPSS Inc. Chicago, Illinois, USA) was used to analyze data. Numerical results are presented as the mean ± standard error, and analysis of variance and unpaired Student’s t-tests were performed to determine the differences between control, PQ, and PQ + PDTC groups at each time point. The Kaplan–Meier method was used to estimate the overall survival time for the three groups. Differences of p < 0.05 were considered significant.

Results

PQ-induced acute toxicity and altered survival rates in experimental groups

The control group displayed a normal biological reaction. In the contrast, PQ treatment group rat started to show the symptoms including restlessness and irritability within 1 h after treatment. Shortness of breath was observed after 2 h of PQ treatment. The respiration and nodding symptoms were most serious at days 1 to 3, along with weight loss, diarrhea, and hematuria. These symptoms improved on days 7 to 28 but became more serious again on days 28 to 56. Seventeen PQ group rats died (2 rats died on days 4, 6, 10, and 41; 3 died on day 5; 4 died on day 12, and 1 died on days 13 and 19) and 9 PQ + PDTC group rats died (3 died on day 5, 3 died on day 6, and 3 died on day 9). Kaplan–Meier survival analysis is reported in Figure 1. The PQ + PDTC group had a higher survival rate than that of the PQ group (p < 0.05).

Figure 1.

Kaplan–Meier survival analysis. Survival analysis results demonstrate a significantly decreased survival rate for the PQ group compared with the PQ + PDTC group (p < 0.05). PQ dramatically induced acute toxicity in these animals during the first 7 days of treatment. PQ: paraquat; PDTC: pyrrolidine dithiocarbamate.

PDTC attenuates PQ-induced pathological changes in lung tissues

Histopathological assessment of the pulmonary tissues was performed on the different experimental groups. Figure 2(a) shows H&E and Masson’s trichrome staining for both the PQ and PQ + PDTC groups. Collagen accumulation in lung tissues was detected by the Masson’s trichrome method ¹⁸ in which blue-stained collagen fibers were observed. Animals from the PQ group showed a significant change in lung tissue structure with alveolar collapse and collagen accumulation due to focal inflammation in the alveolar spaces and septa. No such histological changes or fibrosis were observed in the PQ + PDTC group. According to the lung injury score (Figure 2(b)), the mean score for the PQ + PDTC group significantly decreased starting at day 7 compared to that of the PQ group (p < 0.05).

Figure 2.

Representative images of H&E and Masson’s trichome-stained lung sections and statistical data on injury scores. (a) Images represent micrographs of lung tissue stained with H&E and Masson’s trichrome on days 3, 14, and 56. On day 3, histopathological changes within the PQ group included pulmonary hemorrhage, edema, congestion, and infiltration of inflammatory cells around the bronchia. Only a slight decrease in the alveolar space was found among PQ + PDTC group animals. (b) Pathological scores were significantly decreased in the PQ + PDTC group from day 7 until the end of the experiment, while the PQ group presented higher pathological scores compared with the control group. Original magnification: ×400. *p <0.05: compared with control; ⁁p <0.05: compared with PQ + PDTC group. H&E: hematoxylin and eosin; PQ: paraquat; PDTC: pyrrolidine dithiocarbamate.

Hydroxyproline levels in lung tissues and TGF-β levels in serum

Following 7 days of treatment, the hydroxyproline level in PQ group animals was found to be significantly increased, while this level was decreased in the PQ + PDTC group compared to that of the control group (Figure 3(a)). Meanwhile, the serum TGF-β1 levels were correspondingly increased in PQ-treated animals from day 7 to 56 compared to levels in the control and PQ + PDTC groups (p < 0.05), but no significant difference between control and PQ + PDTC treatment was observed on the final day of the experiment (Figure 3(b)).

Figure 3.

Hydroxyproline and TGF levels in lung tissue and serum. (a) Beginning day 7 of the experiment, PDTC treatment significantly inhibited the hydroxyproline accumulation in lung tissues. (b) ELISA analysis results showed a dramatic decrease in TGF-β1 levels in serum at the beginning of the experiment, suggesting an early TGF response to PQ-induced toxicity in the blood. *p <0.05: compared with control; ⁁p <0.05: compared with PQ + PDTC group. TGF: transforming growth factor; PQ: paraquat; PDTC: pyrrolidine dithiocarbamate; ELISA: enzyme-linked immunosorbent assay.

Protein array results

A NF-κB pathway protein array was performed, and the results revealed that NF-κB pathway proteins contributed to PQ-induced acute lung toxicity (Figure 4(a)). p65, IKKβ, IκB-α, Rel, and TGF receptor 1 levels showed more than a fivefold change compared with the control levels (Figure 4(b)). p65, IKKβ, IκB-α, and TGF receptor 1 expression levels were analyzed by Western blot analysis to verify the array results (Figure 4(c)).

Figure 4.

Protein array results. (a) The protein array sample image represents the Cy3-conjugated protein array signal from the PQ group. (b) Proteins with more than a 1.5-fold change observed in the PQ and PQ + PDTC group animals. (c) Protein expression of four representative NF-κB proteins was verified using Western blot analysis. PQ induced overexpression of these proteins, while PDTC reversed the effect of PQ treatment. PQ: paraquat; PDTC: pyrrolidine dithiocarbamate; NF-κB: nuclear factor κB

Discussion

PQ-induced lung injury in humans and experimental animals is characterized by pro-inflammatory cytokine accumulation and breaks in the alveolar epithelium and fibroblast proliferation. ¹⁹ It is well accepted that PQ achieves these cytotoxic effects by stimulating production of ROS in lung tissue, subsequently leading to an imbalance in the antioxidant system. ^20,21 We found clear indications of morphological injury from days 14 to 56 of PQ administration including alveolar edema, hemorrhage, inflammatory cell infiltration, and swollen alveolar epithelial cells. However, our results also indicate that PDTC protected PQ-exposed lungs from developing fibrosis. Moreover, our data suggest a key role for NF-κB pathway proteins in this protection process.

PDTC is a small molecular thiol compound with antioxidant and anti-inflammatory properties. ¹⁷ It is known that thiol-based antioxidants have the capacity to control and inhibit activation due to a cysteine residue in their side chain. ¹⁹ Hence, recent studies have attracted attention to experimental models that show the inhibition of lung fibrosis by thiol-based antioxidants including N-acetylcysteine, carbocysteine, erdostein, and captopril. ^{22 –24} In this study, PDTC dramatically decreased lung hydroxyproline levels in our experimental PQ + PDTC group compared to levels observed in the PQ group. The presence of hydroxyproline is usually taken as an indication of collagen accumulation; thus, PDTC suppresses collagen accumulation. Our protein array analysis revealed a role for NF-κB in PQ-induced toxicity and the PDTC-mediated protection mechanism. We also showed, using microarray analysis, that PQ significantly elevated NF-κB pathway proteins such as p65, IKKβ, IκB-α, Rel, and TGF receptor 1. Interestingly, the addition of PDTC decreased the progression of fibrosis through effects on these four major NF-κB pathway proteins. The NF-κB pathway is activated under a variety of conditions including infection, exposure to pro-inflammatory cytokines, growth factors, and oxidative stress. ²⁵ Continual expression of TGF-β1 is associated with the development of tissue fibrosis. ²⁶ In xenograft lung fibrosis models, TGF-β1 plays a key role in initiating tissue repair mechanisms and is an important upstream effector of collagen gene expression. ^27,28 The observed inhibitory effect of PDTC on PQ-induced damage and modulation of NF-κB components suggest that PQ-induced fibrosis may be partially blocked by PDTC actions on NF-κB activation, which in turn lead to reduced TGF-β1 gene expression. ²⁹

In conclusion, our results provide evidence that PDTC significantly decreases TGF-β1 levels in the circulatory system and reduces accumulation of collagen deposits in PQ-treated rats in a NF-κB-dependent manner. These findings suggest that PQ-induced pulmonary damage may be reversed by treating with PDTC.