Introduction
Esophageal, gastric and lung cancer are common diseases that pose a serious global threat to health. Chemotherapy agents such as bleomycin, mitomycin and methotrexate may cause pulmonary toxicity, while radiotherapy may lead to radiation pneumonitis (1,2). Lung injury seriously hampers full implementation of treatment, limiting the potential benefits of therapy. The early phase of lung injury is characterized by inflammation (alveolitis), while the late phase is characterized by the organization and deposition of collagen with remodeling (pulmonary fibrosis) (1,2).
The characteristic clinical and histological manifestations of acute lung injury (ALI) are initiated by a well-described network of cytokines (3,4). The acute phase of ALI, characterized by alveolar inflammation, is mediated by tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and transforming growth factor-β (TGF-β) (3,4). In addition, interferon-γ (IFN-γ) is able to maintain the inflammatory response in the lung by inducing macrophages to produce mediators, such as TNF-α, IL-1, IL-6 and IL-8. TNF-α is an important signaling protein that is able to initiate and continually amplify local or systemic inflammatory responses (5).
TGF-β is a key cytokine that induces lung injury and contributes to pulmonary fibrosis, through its actions to induce collagen gene expression or synthesis by stimulation of fibroblast proliferation (3,4,6,7). Expression of the connective tissue growth factor (CTGF) gene acts as a downstream effector of TGF-β1, and is thought to play an important role in pulmonary fibrosis through the promotion of extracellular matrix synthesis (8).
Numerous other signal transduction pathways have been implicated in the pathogenesis of pulmonary fibrosis. For example, the mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathway is essential for the formation of pulmonary fibrosis (9–11). NF-κB is a significant transcription factor that is a key mediator of signal transduction during the acute inflammatory response and pulmonary fibrosis (12,13).
Despite recent advances in our understanding of the epidemiology, pathogenesis and treatment of ALI, this condition remains a significant cause of morbidity and mortality in the critically ill patient population (14). At present, glucocorticoids are the most frequently used anti-inflammatory drugs for the clinical management of ALI (9,15). However, currently there are no approved medical anti-fibrotic therapies (16), and hence the development of effective agents to ameliorate pulmonary fibrosis is urgently needed.
Peiminine is the main component of Fritillaria, and has been used for several years as a traditional Chinese medicine for a variety of conditions including pulmonary fibrosis. Peiminine has been reported to have effects as a relaxant of bronchial smooth muscle and as an antitussive (17,18). In addition, there is evidence that alkaloids isolated from the Fritillaria bulb have anti-inflammatory, as well as antitussive actions (17,18). However, to date there have been no studies exploring whether or not peiminine is able to inhibit pulmonary inflammation and fibrosis.
The aims of this study was to determine whether or not peiminine inhibits lung inflammation and pulmonary fibrosis in rats. Furthermore, the effects of peiminine on lung injury were correlated with changes in the levels of mediators implicated in the pathogenesis of ALI.
Materials and methods
Animals
Age- and gender-matched Sprague-Dawley (SD) rats (weight, 180–220 g) were purchased from the Experimental Animal Center of the Nanjing Medical University, kept in a 12-h dark/light cycle in a temperature- and humidity-controlled room, and fed a standard laboratory diet and water. The experimental procedures were approved by the Animal Care and Use Committee of the Nanjing Medical University, China. Adequate measures were taken to minimize the pain experienced by the experimental animals.
Drugs and reagents
Peiminine (purity >98%) was obtained from Gamma Technology Development Co., Ltd. (Shenzhen China). Dexamethasone (DXS; 0.75 mg) was purchased from Tianjin Tianyao Pharmaceutical Co., Ltd. (Tianjin, China). Bleomycin (8 mg) was obtained from Tianjin Taihe Pharmaceutical Co., Ltd. (Tianjin, China).
Rabbit anti-rat polyclonal antibodies against TGF-β, CTGF, NF-κB, ERK1/2, Fas and FasL were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). IL-4 and TNF-α kits were provided by the Beijing Huaying Biotechnology Institute (Beijing, China). The IFN-γ kit was obtained from the Beijing Huaying Biotechnology Institute, sourced from Adlitteram Diagnostic Laboratories, Inc. (West Palm Beach, FL, USA).
Instruments
The microtome was purchased from Leica (Mannheim, Germany). The optical microscope, Olympus DP71 microscope digital camera and fully automated image acquisition system were obtained from the Olympus Corporation (Tokyo, Japan).
The γ-911 automatic radioimmunoassay (RIA) counter was purchased from the Science and Technology Industrial Company of the China University, and the Stat Fax 2100 automatic microplate reader was purchased from Awareness Technology, Inc. (Palm City, FL, USA).
Experimental groups
Rats were randomly divided into 4 groups: the sham-operated (n=12), the control (n=14), the DXS (n=14) and the peiminine groups (n=10). For the latter 3 groups, intratracheal administration of bleomycin was used to induce lung injury, to allow comparison of the effects of peiminine and DXS. For the sham-operated group, normal saline was applied instead of bleomycin, as the negative control for ALI.
Development of the ALI model in rats
After allowing adjustment to the environment, the rat was anesthetized with chloral hydrate (10%) and fixed on a board in the supine position. For the control, DXS and peiminine groups, bleomycin (5 mg/kg) was instilled into the trachea of the rat using a microliter injector, based on methods described previously in the literature (19). For the sham-operated group, normal saline was administered instead of bleomycin. After intratracheal instillation of bleomycin or saline, the rat was placed in a vertical position and spun for 0.5 min to ensure that the solution was distributed evenly within the lungs.
Administration of drugs
Rats in the sham-operated and control groups were given 5‰ carboxymethyl cellulose sodium (CMC) solution at a dosage of 1 ml/100 g weight; CMC was chosen as its viscosity was similar to that of the drugs used in the other 2 groups. Rats in the DXS group were given an equal volume of DXS solution at a dosage of 0.000405 g/kg weight. Rats in the peiminine group were administered an equal volume of peiminine at a dosage of 0.005 g/kg weight. Drugs were administered daily for 28 consecutive days, using gastric gavage; it has been reported previously that 28 days are required for the formation of lung fibrosis after administration of bleomycin (20).
Alveolitis and pulmonary fibrosis assay
Rats were anesthetized and sacrificed by carotid exsanguination. The left lung was fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) under 15–20-cm H2O pressure. Lungs were embedded in paraffin, and 4-μm sections were prepared. For histology, the sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome. To assay the severity of alveolitis and pulmonary fibrosis, the scoring method described by Szapiel et al(21) was used.
The grading criteria used for alveolitis were: 1 point, no alveolitis; 2 points, mild alveolitis, affecting <20% of the total lung, showing infiltration of mononuclear cells into the widened alveolar septa, and limited to localized regions with involvement of nearby pleural areas; 3 points, moderate alveolitis, affecting an area of 20–50%, with greater pleural involvement; 4 points, severe alveolitis, involving an area >50%, with occasional monocytes in the alveolar space and bleeding caused by consolidation.
The scoring criteria used for fibrosis were: 1 point, no fibrosis; 2 points, mild fibrosis, affecting an area <20% of the whole lung, with fibrosis involving the pleura and subpleural interstitium, and disorders of alveolar structure; 3 points, moderate fibrosis, involving an area of 20–50%, with localized areas of fibrosis extending from the pleura; 4 points, severe fibrosis, involving an area >50%, with fusion of alveolar spaces.
Lung index assay
Rats were anesthetized and sacrificed by carotid exsanguination, and their chest was opened to obtain the lungs. The trachea was removed and discarded, and after drying the surface with filter paper, the lungs were weighed. The lung index was then calculated, based on lung weight and body weight: Lung index = lung weight (g)/body weight (g) ×100%
Assay for inflammatory cytokines
Rats were anesthetized and sacrificed by carotid exsanguination. The blood was collected, and centrifuged at 3,000 rpm for 10 min to obtain serum. The serum was subjected to RIA to determine the levels of IL-4, TNF-α and IFN-γ.
Assay of cell signal transduction pathways
The left lung was fixed with 4% paraformaldehyde in PBS under 15–20-cm H2O pressure. The lung was embedded in paraffin and 4-μm sections were prepared. Immunohistochemistry, using the streptavidin-biotin complex (SABC) method, with calculation of average optical density (IOD), was used to determine the levels of TGF-β, CTGF, NF-κB, ERK1/2, Fas and FasL.
Statistical analysis
Data are expressed as the means ± standard deviation (SD). Statistical analyses were carried out using the SPSS 16.0 software. One-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test were used to compare the results in the various treatment groups. P<0.05 was considered to indicate a statistically significant difference.
Results
General observations
In the initial period after surgical operation, rats in the control group (i.e., with bleomycin-induced ALI) had cold tails and limbs, dark purple tail veins and loss of hair luster. These symptoms gradually receded, showing improvement at 3 days, and had almost disappeared at 7 days. Rats in the control and DXS groups showed a reduced activity, decreased appetite and weight loss. The weight loss was more evident in the DXS group; however, in these two groups, body weight gradually recovered over 18–21 days. In the sham-operated and peiminine groups, no noticeable reduction was observed in activity, appetite or weight. The mortality rates (during the 28-day period) in the control and DXS groups were 1/14 rats. No rats died in the other two groups during this period.
Macroscopic observations of lung tissue
The lung tissue of the sham-operated group was pink, smooth and soft, with good elasticity. However, in the control group, a significant reduction was observed in the amount of normal lung tissue, with increasing occurrence of uneven pale foci, black lesions and reduced elasticity. In the DXS group, the extent of the lesions was smaller compared to the control group, although there was still a clear difference from the sham-operated group. The peiminine group showed no obvious changes, with lung tissue structure resembling that of the sham-operated group.
H&E staining observed under the light microscope
Representative examples of sections from the 4 groups are shown in Fig. 1. The morphological characteristics of the sham-operated group were consistent with those expected of normal lung structure. By contrast, the control group showed widening of the alveolar septa, interstitial edema and inflammatory cell infiltration into the pulmonary interstitium and the alveolar spaces. Although pathological changes were also evident in the DXS and peiminine groups, these lesions were less severe or extensive compared to those observed in the control group.
Masson’s trichrome staining observed under the light microscope
Representative examples of sections from the 4 groups are shown in Fig. 2. In the lung tissue of sham-operated rats, a relatively small amount of collagen fibers was present. In the control group, a substantial increase in the number of collagen fibers was evident, typical of pulmonary fibrosis. Evidence of pulmonary fibrosis was also observed in the DXS and peiminine groups, although to a lesser extent compared to that observed in the control group.
Alveolitis and pulmonary fibrosis scores
As shown in Fig. 3, scores for alveolar inflammation and pulmonary fibrosis were significantly higher in rats in the control group compared to rats in the sham-operated group (P<0.01). Furthermore, the alveolitis and pulmonary fibrosis scores in the DXS and peiminine groups were significantly lower compared to the corresponding scores in the control group (P<0.01).
Lung index as a measure of lung injury in rats
Fig. 4A–C presents data for body and lung weight as well as lung index for the 4 groups. The control group showed a significantly elevated lung index (P<0.01) compared to the sham-operated group, which was associated with a significant increase in lung weight (P<0.05) and a significant decrease in body weight (P<0.01). This increase in the lung index is indicative of bleomycin-induced lung injury in rats of the control group. Furthermore, the peiminine group was found to have a significantly lower lung index (P<0.01), as well as a significantly lower lung weight (P<0.01) compared to the control group. These results suggest that peiminine reduced the extent of the lung injury.
Levels of inflammatory cytokines in the blood
As shown in Fig. 4D–F, 28 days after induction of lung injury, levels of IL-4 (P<0.01) and IFN-γ (P<0.05) were significantly elevated in the control group, compared to the sham-operated group. The levels of TNF-α and IL-4 in the peiminine and DXS groups were not significantly different to the corresponding values in the control group (P>0.05). However, the levels of IFN-γ in the peiminine and DXS groups were significantly lower compared to that of the control group (P<0.01).
Cell signal transduction pathways
As shown in Fig. 5, the levels of TGF-β, CTGF, NF-κB, ERK1/2, FasL and Fas were significantly higher in the control group compared to those in the sham-operated group (P>0.05 for Fas; P<0.01 for the others).
Compared to the control group, the peiminine and DXS groups showed significantly lower levels of TGF-β, CTGF, NF-κB, ERK1/2 and FasL (P<0.01 for all). By contrast, no statistically significant differences were observed in these groups for Fas (P>0.05).
Discussion
The main findings of our study are that peiminine is as effective as DXS in reducing the degree of alveolitis and the extent of pulmonary fibrosis (assessed using histological scoring methods and the lung index), 28 days after bleomycin-induced lung injury in rats. These effects of peiminine were associated with a reduced level of serum IFN-γ, and decreased expression of TGF-β, CTGF, ERK1/2, NF-κB and FasL in lung tissue. The beneficial actions of peiminine may thus be due to the inhibitory effects on these aforementioned mediators, which are known to be involved in the pathogenesis of ALI.
Intratracheal application of bleomycin is a widely used technique for inducing ALI in rodent animal model systems, resulting in an initial development of pulmonary oedema that is followed by a fibrotic interstitial reaction (20,22,23). In the present study, histological comparison of the control (bleomycin) and the sham-operated groups showed clear evidence of inflammation, edema and collagen deposition in lung sections of the control group that were not present in the sham-operated group. Furthermore, the grading of alveolitis and pulmonary fibrosis using established scoring systems demonstrated that bleomycin induced these two pathological changes. In addition, levels of IFN-γ and IL-4 in the serum were significantly increased in the control group (compared to the sham-operated group), as were levels of TGF-β, CTGF, ERK1/2, NF-κB and FasL in lung tissue. These data clearly indicate that bleomycin successfully induced lung injury, alveolitis and pulmonary fibrosis in the rats used in our study, supporting our use of this method as a model of ALI.
Over the past decade, substantial progress has been made in understanding the pathophysiology of lung fibrosis. The design of successful anti-fibrotic therapies may need to focus on mechanisms or pathways, downstream of the inflammatory process, that mediate fibroproliferation. The identification of intracellular signaling pathways eliciting the cellular responses of mesenchymal cell proliferation and differentiation as well as extracellular matrix deposition, may facilitate the development of novel therapeutic approaches to ameliorate the global burden of fibroproliferative diseases (9,24). Inhibition of signal transduction proteins is now widely acknowledged as a valid strategy to combat inflammatory disease (23). Notably, studies have reported that neferine, methyl palmitate, naringin, astragalin, luteolin and paeonol have inhibitory effects on pulmonary fibrosis, due to their actions as anti-inflammatory agents, anti-oxidants and inhibitors of cytokines and NF-κB (22,25–30). Our study suggests that peiminine may also have such beneficial effects, which are comparable to those of DXS.
The process of fibrosis is promoted by early pro-inflammatory mediators, hence blocking of these mediators may be one approach to attenuate fibrosis. Evidence from several clinical studies has indicated that pro-inflammatory cytokines, notably TNF-α, IL-1 and IL-6, participate in the early development of inflammation and play a crucial role in ALI (3,4). TNF-α is known as a primary cytokine, since it amplifies the inflammatory cascade to cause inflammatory injury and recruits neutrophils into the lung (5,23). Furthermore, IL-4 is an anti-inflammatory cytokine that is able to inhibit the function of TNF-α and reduce inflammatory injury to lung tissue (31). In the present study, serum levels of TNF-α and IL-4 were not significantly affected by peiminine and DXS, whereas the level of IFN-γ was reduced. This would suggest that inhibitory actions of peiminine and DXS on alveolitis and pulmonary fibrosis are not secondary to effects on TNF-α and IL-4, but may instead be the consequence, at least in part, of decreased secretion of IFN-γ.
Previous studies have identified a number of chemokines, cytokines and growth factors that mediate pulmonary fibrosis (3,4,7). Of these, TGF-β1 is thought to be one of the key mediators that links inflammation to fibrogenesis. CTGF is a downstream mediator of TGF-β1 that induces connective tissue cell proliferation and extracellular matrix deposition (8). It is therefore of note that, in our experiments, peiminine as well as DXS caused reductions in the tissue expression of CTGF and TGF-β. Upregulation of TGF-β1 and CTGF are known to be critically involved in the pathogenesis of pulmonary fibrosis (23,32). TGF-β1 is a potent pro-fibrotic factor that plays a pivotal role in several pathological processes, including the transition of alveolar epithelial cells to myofibroblasts (33–35). Consistent with this hypothesis, impaired TGF-β responsiveness appears to result in a reduction of fibrosis (32). CTGF has been reported to be useful in diagnosing or predicting disease progression in certain fibrotic diseases (36), while CTGF levels in blood are considered to reflect fibrosis in a variety of organs (37). Furthermore, inhibiting the upregulation of CTGF can attenuate bleomycin-induced ALI and pulmonary fibrosis (23), while certain agents that inhibit bleomycin-induced ALI and pulmonary fibrosis have been reported to act through inhibition of TGF-β1 and CTGF. It is therefore reasonable to conclude that some of the inhibitory effects of peiminine (and also DXS) on bleomycin-induced ALI are via reduced expression of CTGF and TGF-β.
The NF-κB family of transcription factors regulates inflammation, survival, proliferation and other biological processes (12). There are clear links between canonical activation of NF-κB in immune cells to the pathogenesis of inflammatory diseases (13), and the expression of pro-inflammatory mediators is known to be modulated by NF-κB (26). Stimulation of the NF-κB pathway is mediated by diverse signal transduction cascades in response to several stress conditions, such as infection and inflammation. We found that administration of peiminine and DXS were associated with significant reductions in tissue NF-κB expression. These actions may thus contribute to the protective effects of these agents on ALI.
The MAPK-ERK signaling cascade is a major pathway controlling cellular processes associated with fibrogenesis, including growth, proliferation and survival. In progressive pulmonary fibrosis associated with increased MAPK/ERK activation, ERK has been reported to be primarily activated in the mesenchymal cells of the fibrotic lesions (9). Clinical findings have demonstrated an upregulation of MAPK/ERK in human fibrotic disease (10). The MAPK/ERK pathway is a logical target for potential fibrosis therapy, as several fibrogenic cytokines signal through MAPK/ERK (11), and selective inhibition of MAPK prevents the development and attenuates the progression of fibrosis, when administered as a rescue therapy. Our findings that peiminine and DXS cause reduced expression of ERK1/2 in lung tissue indicate that decreased signaling through the MAPK/ERK pathway contributes to the anti-fibrotic effects of these drugs. In addition, actions on FasL, which is also involved in the fibrotic process (38), may also play a role in the effects of peiminine and DXS.
Our study is not without limitations. First, although we have shown that peiminine has protective effects against bleomycin-induced ALI, it cannot be certain that such effects would extend to other chemotherapy agents or to radiation-induced injury. However, the bleomycin model is widely used and validated, hence our data are likely to have applicability to human patients, at least to a certain extent. Second, although changes in the levels of various cytokines and mediators have been identified following peiminine treatment in our rat model system, the primary mediators that contribute to the beneficial actions of peiminine are yet to be identified. In addition, it cannot be definitively concluded that similar changes in mediator levels would be seen in human patients. Additional studies are required to expand upon our observations, and describe the mechanisms underlying the actions of peiminine more precisely.
In conclusion, our findings indicate that peiminine has beneficial effects protecting against bleomycin-induced lung injury in rats, and that these effects are comparable to those of DXS. Furthermore, the attenuation of pulmonary fibrosis by peiminine is associated with a reduction in the levels of IFN-γ in the blood, and CTGF, TGF-β, NF-κB, ERK1/2 and FasL in lung tissue. Thus, our findings provide evidence that peiminine may have therapeutic potential in the treatment of ALI and pulmonary fibrosis.