Abstract
Recurrent primary spontaneous pneumothorax (PSP) is a troublesome problem and a major concern for the patients. This study examined whether nuclear factor erythroid 2‐related factor 2 (Nrf2) expression in alveolar type I pneumocytes was associated with the clinical manifestations of PSP patients including disease recurrence. Eighty‐eight PSP patients who were managed with needlescopic video‐assisted thoracoscopic surgery (NVATS) were included in this study. Immunohistochemistry (IHC) was assessed to determine Nrf2 expression in resected lung tissues and the results were correlated with clinicopathological characteristics by the chi‐square or the Fisher's exact test. The prognostic value of Nrf2 for overall recurrence was evaluated by univariate and multivariable Cox regression model. The expression of Nrf2 was observed in type I pneumocytes of lung tissues from PSP patients by IHC. We found that low Nrf2 expression in PSP patients, especially in young (age ≤ 20, p = 0.033) and body mass index (BMI) ≥18 kg/m2 (p = 0.019) groups, was significantly correlated with PSP recurrence. In the univariate and multivariate analyses, high Nrf2 expression was a significant protective factor for overall recurrence in PSP patients (univariate: p = 0.026; multivariate: p = 0.004). The expression level of Nrf2 in alveolar type I pneumocytes was a potential factor involved in PSP recurrence. Our findings suggest that elevated Nrf2 expression in PSP patients may be a promising way for reducing PSP recurrence.
Keywords: Nrf2, Pneumothorax, Recurrence, Type I pneumocyte
Introduction
Primary spontaneous pneumothorax (PSP) is a clinical problem in younger populations and its occurrence rate is approximate 18–28/100,000 per annum in males and 1.2–6.0/100,000 per annum in females [1]. PSP is present without trauma or precipitating cause and arises in patients without underlying pulmonary diseases. The incidence of recurrence rate is about 30%, ranging 16–52% and the majority of recurrence occurs in 6 months to 2 years [[2], [3], [4]].
PSP occurs when subpleural blebs or bullae are ruptured, due to inflammation, hypoxia, and oxidative stress [[5], [6], [7]]. Previous studies reported that inflammatory changes and oxidative stress in the pleural cavity might be involved in the formation of emphysema‐like changes (ELCs) in PSP patients [5]. The ELCs in PSP is related to elastolysis which is induced by an imbalance between oxidants and antioxidants [8]. In these processes, type I pneumocytes play a key role in the oxidative stress response [[9], [10]].
Nuclear factor erythroid 2‐related factor 2 (Nrf2) is the transcription factor that belongs to the Cap'n'collar (CNC)‐basic region leucine zipper (bZIP) family [[11], [12]]. Recent studies have indicated that Nrf2 is the regulator of redox homeostasis and inflammatory response [[13], [14]]. It plays a cytoprotective role in detoxification and chemoprevention through the activation of phase II gene expression [[15], [16]]. Previous studies reported that Nrf2 and its target genes (heme oxygenase 1, NADPH quinone oxidoreductase, and biliverdin reductase), decreased hyperoxia‐induced acute lung injury and were associated with alveolar macrophage oxidative stress in smoking patients with PSP [[7], [17]].
Most of the internal surface area of the lung is covered by type I pneumocytes [[18], [19]]. However, Nrf2 expression in type I pneumocytes of PSP and its association with PSP recurrence have not been reported previously. In the present study, the expression pattern of Nrf2 in type I pneumocytes of PSP was evaluated by immunohistochemistry (IHC) and its association with clinical variables and overall recurrence was further analyzed.
Materials and methods
Study groups
This retrospective study included 88 PSP patients with age ≤40 years diagnosed by chest radiography and computed tomography (CT) scan at our institution, from January 2012 through December 2013. Cases with a secondary pneumothorax, clinical lung disease (i.e., COPD, malignancy, asthma, pneumonia), or pulmonary surgery (i.e., lobectomy, wedge resection of the lung) were excluded from this study. Indications for surgery included recurrence, persistent air leak (for ≥5 days), or patient preference. Radiologic proof of relapse of pneumothorax after first episode of PSP was defined as recurrence group. Patients for surgical treatment at their first admittance with PSP and a 2‐year follow‐up without relapse were defined as non‐recurrence group. The age, sex, body mass index (BMI), smoking status, and side of pneumothorax were recorded. All the specimens with blebs and adjacent normal lung tissues were obtained while patients were undergoing needlescopic video‐assisted thoracoscopic surgery (NVATS) with an endoscopic linear stapler described previously [20]. This study was approved by the Institutional Review Board (KMUH‐IRB‐20130268) and informed consent was obtained from all participants.
Immunohistochemistry
IHC staining for Nrf2 in lung tissues of 88 patients was performed on a Bond‐Max autostainer (Leica Microsystems, Bannockburn, IL, USA) as described previously [21]. In brief, resections were incubated with Nrf2 (1:200 dilution, GeneTex, GTX103322, Irvin, CA, USA) antibody and color detection was performed with DAB (3,3′‐diaminobenzidine tetrahydrochloride). To confirm that the cell type stained positive for Nrf2 was indeed type I pneumocyte, an adjacent section to that stained for Nrf2 was stained with a monoclonal antibody recognizing D2‐40 marker on type I pneumocytes (D2‐40, 1:100 dilution, ThermoFisher, MA1‐83884, Waltham, MA, USA) [22]. Slides were counterstained with hematoxylin and images of IHC‐stained sections were captured by a Nikon Eclipse Ti microscope (Tokyo, Japan) and processed by Nikon NIS‐Elements Version 4.30 software (Tokyo, Japan). Negative control was performed in parallel with the IHC procedure with non‐addition of Nrf2 antibody.
Slide evaluation
Nrf2 staining was scored according to the Quick score (Q‐score) method [[23], [24]], which was based on the intensity and the percentage of stains, and determined by two independent experts who were blinded to clinical outcome under the same conditions. Four visual fields were evaluated randomly and the average Q‐score in the four fields was calculated for each slide. The staining intensity was stratified into quartiles (0, absent; 1, weak; 2, moderate; 3, strong) and the staining percentage was scored as: 0 (0% positive cells), 1 (≤25% positive cells), 2 (26–50% positive cells), 3 (51–75% positive cells), or 4 (≥76% positive cells). The sum of intensity and percentage scores is the Q‐score of a tissue section ranging from 0 to 7. Score of ≤5 was categorized as low expression, while score of >5 was categorized as high expression for statistical analysis.
Statistical analysis
The associations of high and low Nrf2 expression with age, gender, BMI, recurrence, smoking status, and side involved were analyzed by the chi‐square test or the Fisher's exact test. Hazard ratio (HR) and 95% confidence interval (CI) from Cox proportional hazard regression models were used in univariate and multivariate analysis to evaluate associations between overall recurrence and clinicopathological parameters. Statistical analyses were performed using the SPSS software (SPSS Inc, Chicago, IL, USA) and p values <0.05 were considered statistically significant.
Results
Expression level of Nrf2 in alveolar type I pneumocytes of PSP patients
A total of 88 PSP patients, 76 males and 12 females, were included in this study. The mean age of the patients was 21.0 ± 5.2 years and 45 patients presented with recurrence of pneumothorax. The information for BMI, smoking status, and lesion location is summarized in Table 1. Nrf2 was predominantly expressed in PSP lung tissues but not in the adjacent normal lung tissues (Fig. 1). In this study, D2‐40 monoclonal antibody was applied to recognize type I pneumocytes and confirm that Nrf2‐stained cells were indeed type I pneumocytes (Fig. 1E). The expression status of Nrf2 in type I pneumocytes of pneumothorax tissues was classified into low and high expression (score ≤ 5 or >5) according to the Q‐score method described in Materials and Methods. As shown in Table 2, 26 (29.5%) cases of the PSP tissues exhibited high Nrf2 expression, while 62 (70.5%) cases of the PSP tissues exhibited low Nrf2 expression.
Table 1.
Clinical characteristics of patients with primary spontaneous pneumothorax.
| Characteristics | n = 88 |
|---|---|
| Age, years, mean ± SD | 21.0 ± 5.2 |
| Sex, n (%) | |
| Male | 76 (86.4%) |
| Female | 12 (13.6%) |
| Height, cm, mean ± SD | 172.6 ± 7.5 |
| Body weight, kg, mean ± SD | 56.3 ± 8.8 |
| BMI, kg/m2, mean ± SD | 18.8 ± 2.2 |
| Smoking, n (%) | |
| Yes | 17 (19.3%) |
| No | 71 (80.7%) |
| Side involved, n (%) | |
| Unilateral | 63 (71.6%) |
| Bilateral | 25 (28.4%) |
| Recurrence, n (%) | |
| Yes | 45 (51.1%) |
| No | 43 (48.9%) |
Data are presented as the mean ± SD and n (%).
BMI = body mass index; SD = standard deviation.
Figure 1.
Immunohistochemical staining for low expression (A) and high expression (B) of Nrf2 in alveolar type I pneumocytes (arrow) of PSP. PSP lung tissue slides were stained using IHC methods descripted in Materials and methods. Nrf2 expression in the matched adjacent normal lung tissue (C) and negative control (D) were also shown. (E) Adjacent section next to the section stained for Nrf2 in (A) was stained for type I pneumocyte marker, D2‐40. The representative photographs were shown with 400× magnification. IHC = immunohistochemistry; Nrf2 = nuclear factor erythroid 2‐related factor 2; PSP = primary spontaneous pneumothorax.
Table 2.
Clinical characteristics of pneumothorax patients with Nrf2 expression.
| Variables | Item | Patient No. (%) | Low | High | p value | ||
|---|---|---|---|---|---|---|---|
| No. | % | No. | % | ||||
| Age, years | ≤20 | 51 (58.0) | 33 | 64.7 | 18 | 25.3 | 0.165 a |
| >20 | 37 (42.0) | 29 | 78.4 | 8 | 21.6 | ||
| Sex | Male | 76 (86.4) | 54 | 71.1 | 22 | 28.9 | 0.743 b |
| Female | 12 (13.6) | 8 | 66.7 | 4 | 33.3 | ||
| BMI, kg/m2 | <18 | 35 (39.8) | 26 | 74.3 | 9 | 25.7 | 0.522 a |
| ≥18 | 53 (60.2) | 36 | 67.9 | 17 | 32.1 | ||
| Recurrence | No | 43 (48.9) | 25 | 58.1 | 18 | 41.9 | 0.013 a |
| Yes | 45 (51.1) | 37 | 82.2 | 8 | 17.8 | ||
| Smoking status | No | 71 (80.7) | 48 | 67.6 | 23 | 32.4 | 0.375 b |
| Yes | 17 (19.3) | 14 | 82.4 | 3 | 17.6 | ||
| Side involved | Unilateral | 63 (71.6) | 46 | 73.0 | 17 | 27.0 | 0.403 a |
| Bilateral | 25 (28.4) | 16 | 64.0 | 9 | 36.0 | ||
BMI = body mass index; Nrf2 = nuclear factor erythroid 2‐related factor 2.
The p value was calculated by the chi‐square test.
The p value was calculated by the Fisher's exact test.
Association between Nrf2 expression and clinicopathological parameters of PSP patients
Nrf2 expression in PSP lung tissues was correlated with age, sex, BMI, recurrence, smoking status, and side involvement (Table 2). High Nrf2 expression in type I pneumocytes of PSP lung tissues was significantly associated with the decreased recurrence risk (p = 0.013). Age, sex, BMI, smoking status, and lesion location were not correlated with Nrf2 expression. Further analysis in young (age ≤ 20 years) PSP patients revealed that decreased PSP recurrence was associated with high Nrf2 expression in type I pneumocytes (p = 0.033) (Table 3). A similar result was observed in BMI ≥18 kg/m2 groups (p = 0.019) (Table 4).
Table 3.
Clinical characteristics of young (age ≤ 20) pneumothorax patients with Nrf2 expression.
| Variables | Item | Patient No. (%) | Low | High | p value | ||
|---|---|---|---|---|---|---|---|
| No. | % | No. | % | ||||
| Sex | Male | 47 (92.2) | 31 | 66.0 | 16 | 34.0 | 0.607 b |
| Female | 4 (7.8) | 2 | 50.0 | 2 | 50.0 | ||
| BMI, kg/m2 | <18 | 24 (47.1) | 16 | 66.7 | 8 | 33.3 | 0.782 a |
| ≥18 | 27 (52.9) | 17 | 63.0 | 10 | 37.0 | ||
| Recurrence | No | 21 (41.2) | 10 | 47.6 | 11 | 52.4 | 0.033 a |
| Yes | 30 (58.8) | 23 | 76.7 | 7 | 23.3 | ||
| Smoking status | No | 47 (92.2) | 31 | 66.0 | 17 | 34.0 | 1.000 b |
| Yes | 4 (7.8) | 3 | 75.0 | 1 | 25.0 | ||
| Side involved | Unilateral | 35 (68.6) | 24 | 68.6 | 11 | 31.4 | 0.393 a |
| Bilateral | 16 (31.4) | 9 | 56.2 | 7 | 43.8 | ||
BMI = body mass index; Nrf2 = nuclear factor erythroid 2‐related factor 2.
The p value was calculated by the chi‐square test.
The p value was calculated by the Fisher's exact test.
Table 4.
Clinical characteristics of BMI ≥18 kg/m2 pneumothorax patients with Nrf2 expression.
| Variables | Item | Patient No. (%) | Low | High | p value | ||
|---|---|---|---|---|---|---|---|
| No. | % | No. | % | ||||
| Age, years | ≤20 | 27 (50.9) | 17 | 63.0 | 10 | 37.0 | 0.430 a |
| >20 | 26 (49.1) | 19 | 73.1 | 7 | 26.9 | ||
| Sex | Male | 46 (86.8) | 32 | 69.6 | 14 | 30.4 | 0.667 b |
| Female | 7 (13.2) | 4 | 57.1 | 3 | 42.9 | ||
| Recurrence | No | 25 (47.2) | 13 | 52.0 | 12 | 48.0 | 0.019 a |
| Yes | 28 (52.8) | 23 | 82.1 | 5 | 17.9 | ||
| Smoking status | No | 44 (83.0) | 29 | 65.9 | 15 | 34.1 | 0.701 b |
| Yes | 9 (17.0) | 7 | 77.8 | 2 | 22.2 | ||
| Side involved | Unilateral | 35 (66.0) | 25 | 71.4 | 10 | 28.6 | 0.446 a |
| Bilateral | 18 (34.0) | 11 | 61.1 | 7 | 38.9 | ||
BMI = body mass index; Nrf2 = nuclear factor erythroid 2‐related factor 2.
The p value was calculated by the chi‐square test.
The p value was calculated by the Fisher's exact test.
Correlation between Nrf2 expression levels in PSP tissues and overall recurrence in patients
To evaluate the risk factors for overall recurrence in pneumothorax patients, univariate and multivariable Cox proportional hazard regression models were applied (Table 5). In the univariate analysis, high Nrf2 expression in type I pneumocytes significantly associated with a decreased risk for recurrence (HR = 0.42, 95% CI = 0.20–0.90, p = 0.026). Further multivariate analysis also identified that high Nrf2 expression was significantly correlated with low overall recurrence (HR = 0.30, 95% CI = 0.13–0.67, p = 0.004), while age, sex, BMI, smoking status, and side involved were not risk factors for pneumothorax recurrence (Table 5).
Table 5.
Univariate and multivariable analysis for overall recurrence in pneumothorax patients.
| Variables | Item | Univariate | Multivariable | ||||
|---|---|---|---|---|---|---|---|
| HR | 95% CI | p value | HR | 95% CI | p value | ||
| Age, years | >20 | 0.57 | (0.32, 1.01) | 0.056 | 0.50 | (0.25, 1.02) | 0.056 |
| ≤20 | 1.00 | 1.00 | |||||
| Sex | Female | 1.27 | (0.58, 3.01) | 0.589 | 1.97 | (0.79, 4.89) | 0.146 |
| Male | 1.00 | 1.00 | |||||
| BMI, kg/m2 | ≥18 | 1.05 | (0.57, 1.90) | 0.885 | 1.19 | (0.62, 2.25) | 0.605 |
| <18 | 1.00 | 1.00 | |||||
| Smoking status | Yes | 0.50 | (0.21, 1.19) | 0.117 | 0.55 | (0.22, 1.40) | 0.211 |
| No | 1.00 | 1.00 | |||||
| Side involved | Unilateral | 1.27 | (0.67, 2.42) | 0.463 | 1.45 | (0.73, 2.88) | 0.285 |
| Bilateral | 1.00 | 1.00 | |||||
| Nrf2 | High | 0.42 | (0.20, 0.90) | 0.026∗ | 0.30 | (0.13, 0.67) | 0.004∗ |
| Low | 1.00 | 1.00 | |||||
*p < 0.05, statistically significant.
BMI = body mass index; CI = confidence interval; HR = hazard ratio; Nrf2 = nuclear factor erythroid 2‐related factor 2.
Discussion
PSP is a critical clinical problem and several reports have suggested early surgery to decrease the recurrence rate and repeated admission [[25], [26]]. The recurrence risk is about 25% after the first pneumothorax episode and about 70% after the second episode if without adequate managements [[2], [27]]. In this study, all the PSP patients received NVATS. Our previous study compared the NVATS with the conventional video‐assisted thoracic surgery (CVATS) for PSP patients [28]. The operative time was shorter and intraoperative blood loss was less in the NVATS group. The postoperative pain in visual analog scale was also significantly less in the NVATS group. There were three postoperative recurrences in the NVATS group (3 out of 106) while one was observed in the CVATS group (1 out of 89), which was statistically insignificant. Based on our previous study, different surgical approaches do not influence the outcome of PSP recurrence. Moreover, the pulmonary high‐resolution CT findings of blebs or bullae have been used to predict the risk of pneumothorax recurrence but with controversial results [29]. While one study reported that greater CT‐based results were associated with higher recurrence risks in PSP patients [30], another study showed no significant association between CT‐based results and recurrence rates [31]. Until now, there is no available factor correlated with PSP recurrence after different interventions. In this study, we found that the adjusted hazard ratio for overall recurrence was decreased in PSP patients with high Nrf2 expression (Table 5). To our knowledge, the correlation of Nrf2 with recurrence of PSP has not been previously documented.
Nrf2‐regulated redox balance has been reported in some airway diseases [[32], [33]]. However, there has been no evidence investigating Nrf2 expression in PSP lung tissue or its association with clinical parameters of PSP patients. Several factors, such as smoking, age, or BMI, have been found to be involved in pneumothorax recurrence [2]. Oxidative stress was reported to be involved in the predisposing effects of smoking on PSP [34]. Nrf2 plays a protective role in oxidative airway diseases by expressing antioxidant response element‐regulated genes [[16], [35]]. However, the present study showed no significant association between smoking and Nrf2 expression level in PSP patients. In fact, the negative result is the small sample size of smoking patients with PSP (17 out of 88) included in our study. Further large scale clinical studies are required to determine whether the expression levels of Nrf2 could be a cytoprotective factor against oxidative stress response in smoking patients with PSP. Nevertheless, in the current study, Nrf2 was expressed in type I pneumocytes, which are essential for maintenance of normal alveolar homeostasis and protection from injury.
In agreement with previous studies, we observed that some PSP patients had lower BMI (<18 kg/m2) (Table 2). However, there was no significant correlation between BMI and the expression level of Nrf2 in PSP patients. Further analysis in patients with BMI ≥18 kg/m2 but not in patients with BMI <18 kg/m2 revealed that increased recurrence rate was correlated with low Nrf2 expression (Table 4). In this study, we also found that most of the PSP patients were young. Analysis in young subjects revealed that the recurrence rate was significantly different between low and high Nrf2 expression groups (Table 3). PSP frequently recurs in young patients who are expected to have normal physical activity. Therefore, evaluation of the protective factor for PSP recurrence, including the analysis of Nrf2 expression in young PSP patients, may provide useful information in medical practice. Together, the BMI (≥18 kg/m2), age (≤20 years), and Nrf2 expression levels may be the confounder in prediction of PSP recurrence. Further large‐scale clinical cases are required to enforce the findings in this study.
Of note, the recurrence rates were high in the current study, with 51.1% of patients having PSP recurrence after the first episode of pneumothorax. One reason for the high recurrence rate in our study was that most of the recurrent patients were primarily treated at nearby local hospitals and were referred to our medical center for surgical management.
In conclusion, high expression level of Nrf2 is a protective factor for PSP recurrence. Further multi‐center clinical studies are required to solidify our finding and address its clinical application in the future.
Acknowledgments
This study was supported by grants from the Kaohsiung Medical University Hospital (KMUH102‐2M19, KMUH102‐2M21), Kaohsiung Medical University (KMU‐M106028), and Kaohsiung Municipal Ta‐Tung Hospital (kmtth‐104‐013) of Taiwan.
Conflicts of interest: All authors declare no conflicts of interest.
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