3.3 Comparison of CT changes between groups
The CT image changes after 10 days are shown in Table 4. There was no difference in the number of patients with imaging absorption ≥50% between the groups. From the CT score changes, the NAC group did not show a greater improvement in CT scans than the non-NAC group.
The pathophysiological mechanisms of CAP include microorganism invasion, airway damage, and activation of the immune defense systems. Polymorphonuclear neutrophils and macrophages kill these microorganisms by using ROS and lysosomal enzymes, including proteinases. The increase in ROS concentration and proteolytic enzymes may be reflected at the systemic level as an increment in the oxidative stress and airway remodeling biomarkers. Oxidative stress takes part in host innate immune response to foreign pathogens, and increases the production of mediators of pulmonary inflammation.
Numerous studies have shown that there was a higher oxidative stress in CAP patients compared with healthy volunteers. The effects of oxidative stress in the airway as well as in other organs depend on ROS concentration and time of exposure. In general, higher levels of ROS produce damage in biomolecules (e.g., lipid peroxidation) and induce intracellular signaling pathways leading to cell death, mainly through apoptosis. Secondly, the impairment of the antioxidant capacity in CAP could also enhance cell damage. CAP patients presented greater lipid peroxidation, and antioxidant status alterations correlated with clinical severity.
In a way, weakening the oxidative stress may mitigate organ damage. In our study, there was a more significant decrease in plasma MDA level and a more considerable rise in plasma TAOC level after treatment in the NAC group, compared with those in the non-NAC group. These showed that NAC had increased the protective markers for oxidative stress. Besides, with NAC treatment, the TNF-α level decreased more in the NAC group than in the non-NAC group. It was reflected that NAC treatment could protect lung tissue by alleviating oxidative stress and diminishing inflammatory factors such as TNF-α.
N-acetylcysteine, a glutathione precursor, can replenish the total combined thiols (cysteine, cysteinylglycine, glutathione and homocysteine), interact with the electrophile groups of ROS, and then raise the total anti-oxidant capacity. NAC protects alveolar type II cells against injury induced by cigarette smoke in knockout mice lacking the nuclear factor erythroid 2-related factor-2 (Nrf2), which is a redox-sensitive transcription factor and is a key regulator of the antioxidant defense system. NAC pretreatment effectively prevented thiobarbituric reactive substances accumulation, lung edema, and polymorphonuclear neutrophil (PMN) influx into the lungs induced by concentrated ambient thiobarbituric particles. Previous studies have demonstrated the potential antioxidant, anti-inflammatory and mucolytic properties of NAC in COPD. Addition of NAC to the standard treatment of COPD exhibited beneficial effects in disease exacerbations, symptom improvement, and a decline in oxidative stress parameters. High dose NAC improves clinical outcome of COPD exacerbation patients by ameliorating oxidative stress and inflammatory response thereby improving lung spirometry and pulmonary oxygenation.
Besides, NAC meets the need by virtue of its anti-inflammatory action. Study indicates that IL-8, IL-6, and TNF-a could be strongly inhibited by NAC at the expression and release level in alveolar type II cells infected with influenza virus A and B and respiratory syncytial virus. NAC inhibited the activation of NF-κB in alveolar macrophages induced by TNF-α, and was an effective inhibitor of TNF-α/IL-1 β-stimulated intercellular adhesion molecule-1 (ICAM-1) and IL-8 release in endothelial and epithelial cells.
Research suggests the effects of NAC differ in vivo and in vitro and are highly dose-dependent. In vitro anti-inflammatory effects were seen at high but not at low concentrations. NAC administered at high concentrations significantly inhibited the release of IL-1β, IL-8, and TNF-α induced by lipopolysaccharide (LPS) incubation in an ex vivo model of COPD exacerbation. But, on the other hand, some long-term effectiveness is reported in several in vivo studies even at low doses. The lower doses of NAC given in vivo might require longer time in order to achieve sustained effects on the cellular thiols which lead to changes in the redox status.[34,35] This showed that high dose NAC (1200 mg/d) significantly decreased IL-8 levels after 10 days of treatment in patients with COPD exacerbations. In our test, administration with NAC (1200 mg/d) for 7 days could significantly decreased TNF-α levels in CAP patients.
Most studies have shown NAC increased the SOD level or activity in lung injury. On the contrary, in our research, addition of NAC did not provide more improvement in SOD activity compared with conventional therapy, which was in accord with Forgiarini study. Nagata research showed that NAC increased the protein and mRNA expression of MnSOD without altering the mRNA expression of other antioxidant enzymes, including GPx1 (glutathione peroxydase 1), CuZnSOD, and extracellular SOD (ecSOD). Three isoforms of SOD have been found in mammalian cells: CuZnSOD, MnSOD, and ecSOD. We suppose that the effect of NAC on SOD might be determined by the kind of SOD, detecting time, and some other factors.
There was no difference in the improvement of pneumonia as assessed by CT between NAC group and non-NAC group. This might be because the observation period was not long enough. In addition, the pneumonia image absorption time could be influenced by the etiology, host factors, immune factors, and some other factors. So further investigation is needed to reveal any difference in clinical outcome between the 2 patient groups.
Different results might be found by alternative methods of administering NAC. Inhalation of NAC solution can directly act on the airway, so that it might come into effect rapidly, with high bioavailability. Yoshito research showed that ex vivo treatment of donor lungs with inhaled NAC reduced inflammatory response via its antioxidant activity in experimental porcine lung transplantation. Study of NAC inhalation in ventilator-associated pneumonia showed that with prolonged mechanical ventilation, biofilm structure improved, biofilm culture positive rate and incidence of ventilator-associated pneumonia decreased. Tomioka pilot study indicated that aerosolized NAC (352 mg/d) for idiopathic pulmonary fibrosis may delay disease progression. In the literature we referred to when we planned this study in 2016, most studies had used NAC by oral administration in antioxidant study of patients with COPD and influenza.[3,8,9,11,29,41] Therefore oral administration was chosen in this study but on further consideration, using NAC solution by inhalation might be a better choice. The effect of inhalation of NAC solution on oxidative stress and inflammatory factors in respiratory diseases is an interesting subject for future study.
This study has some limitations. The major one being the small sample size. This insufficiency could reduce the test power, limit the ability of our study to generalize, and prevent adjustment for confounding effects. While in some similar studies, the sample number is not large either. De Backer et al studied 12 patients to study the effect of high-dose N-acetylcysteine on airway geometry, inflammation, and oxidative stress in COPD patients. Yuanyuan Chen's research showed Vitamin C mitigated oxidative stress and proinflammatory mediator in severe CAP, with 15 patients in each group. Trefler used small sample in a pilot study to show oxidative stress in immunocompetent patients with severe community-acquired pneumonia. For all that, it is suggested that the study with large sample if possible with patients from multiple centers may have stronger test efficiency, and provide a more convincing conclusion.
Another limitation is that the study did not record details of the etiologic agent. Patients in our study were diagnosed with bacterial community-acquired pneumonia according to ATS/IDSA Guidelines. For the pathological features, treatment and disease course of tuberculosis and fungus infection are different from those of bacterial pneumonia, patients considered to have tuberculosis or fungus infection were not included. Primary viral pneumonia was defined in patients presenting during the acute phase of influenza virus illness with acute respiratory disease and unequivocal alveolar opacification involving ≥2 lobes with negative respiratory and blood bacterial cultures. There might be some differences in oxidative stress status between bacterial and viral pneumonia. Primary viral pneumonia cases were not admitted to the study either.
It has been reported that different microorganisms may elicit different cytokine activation patterns in CAP. The lowest inflammatory expression was found in unknown cause and the highest was found in Legionella pneumophila, Streptococcus pneumoniae, and Enterobacteriaceae.Atypical bacteria exhibit an inflammatory pattern closer to that of viruses. In our study, although viral pneumonia, tuberculosis, and fungus infection were not included, we did not make further stratification in patients selected on the basis of pathogen, which might likely reveal more details in changes of oxidative stress and inflammatory cytokines, and this is also likely to lead to bias.
The study showed no difference in the improvement of CT between NAC group and non-NAC group. One reason for this might be the observation period was not long enough. This is also one of the limitations.
We included patients consecutively in this study. For various reasons, some patients who were selected at the beginning were excluded or signed out halfway. Some factors such as age, sex, body mass index, smoking habits, comorbidities, drugs, and pathogen, may have impact on the oxidative stress and affect the equilibrium between groups. So, the patients who had very advanced age (≥70 years old), severe obesity, heavy smoking, or other sever systemic diseases were not admitted into the study groups. The levels of oxidative stress and inflammatory cytokines in severe pneumonia differ from those in non-severe pneumonia. Thus, we chose patients with PSI score I–III for allocation into the study groups. There might be difficulties in collecting patients who have pneumonia alone without other comorbidities within a certain time period. Thus, there could be potential bias, and this is one of the reasons for the small sample size. Although we have taken some measures, for instance, stratified randomization and some exclusion methods mentioned above, selection bias still existed.
In our study, we did not make stratification on the basis of pathogens, and the confounding effect that pathogens may have had on the 2 groups was not adjusted for. Therefore, this is also likely to be one cause for bias.
In addition, we did not make this a double-blind study. There might be interference from observers’ subjective factors. This is another likely reason for bias.
Therefore, we consider that a further study using a large sample over a longer research period in pneumonia patients with different etiologies and severity may reveal more details of the effect of antioxidant treatment.
In summary, we demonstrated that CAP exhibited significant increase of oxidative stress. Addition of NAC therapy to the CAP patients reduced MDA and TNF-α and increase TAOC more than standard therapy alone. Thus, our study suggests that NAC inhibited oxidative stress and reduced the inflammatory factors in pneumonia. Treatment with antioxidants NAC might reduce oxidative and inflammatory damage in pneumonia patients.
Investigation: Qianwen Zhang, Yuanrong Ju, Yan Ma, Tao Wang.
Methodology: Qianwen Zhang, Yuanrong Ju, Yan Ma, Tao Wang.
Resources: Qianwen Zhang.
Validation: Qianwen Zhang.
Visualization: Qianwen Zhang.
Writing – original draft: Qianwen Zhang, Yuanrong Ju, Yan Ma, Tao Wang.
Writing – review & editing: Qianwen Zhang, Yuanrong Ju, Yan Ma, Tao Wang.
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Keywords:Copyright © 2018 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.
N-acetylcysteine; oxidative stress; pneumonia