Effect of a combination of interferon-α and ambroxol on children with pneumonia, and on serum amyloid A: A randomized controlled study

Purpose: To investigate the effect of combination of interferon-a with ambroxol on children with pneumonia, and its effect on serum amyloid A (SAA). Methods: A total of 140 children who presented with pneumonia in Cangzhou Central Hospital from January 2019 to November 2020 were selected and randomly assigned to control group and study group. All the children were treated with conventional therapy against cough and phlegm, anti-infection, oxygen inhalation, antipyretic, water and electrolyte correction, and nutritional support. The control group received aerosol inhalation of ambroxol + physiological saline, in addition to conventional treatment. The study group was given aerosol inhalation of recombinant human interferon-α1b + ambroxol + physiological saline. The two groups were treated continuously for 7 days. The time taken for clinical symptoms to subside in each group, as well as serum levels of amyloid A (SAA), interleukin 6 (IL-6), immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG) before treatment and 7 days post-treatment were evaluated. Clinical efficacy and incidence of adverse reactions after 7 days of treatment were also obtained and compared. Results: The time taken for symptom disappearance was significantly shorter in the study group than in control group, and inflammation indices were lower in the study group than in control group (p < 0.05). Levels of IgA, IgM, and IgG in the two groups after treatment were significantly elevated, but they were markedly higher in the observation group than in control group (p < 0.05). Clinical treatment efficacy was superior in the study group, relative to control (p < 0.05). However, no statistical disparity in incidence of adverse reactions was witnessed between the two groups (p > 0.05). Conclusion: A combination of interferon-α1b and Ambroxol may be a boon in the treatment of children with pneumonia due to its efficacy and effect on inflammatory response and SAA levels.


INTRODUCTION
Pediatric pneumonia is one of the commonest pediatric diseases. It occurs predominantly in infants and young children, due to factors such as pathogen infection or inhalation of amniotic fluid and oil, as well as allergic reactions and other inflammatory reactions in the lungs. It manifests mainly as fever, cough, pulmonary wet rales, difficulty in breathing and shortness of breath, and it is considered as one of the deathrelated diseases in infants and young children [1,2]. The targets used for controlling pediatric pneumonia include infection controls, relief of symptoms, correction of hypoxia, and prevention of progression of the disease [3]. Ambroxol is a frequently used clinical expectorant which substantially improves respiratory tract function [4].
Definite outcomes of the use of recombinant human interferon-α1b in pediatric pneumonia have been widely documented [5]. However, not much is known about the effect of combined use of recombinant human interferon-α1b and Ambroxol on pediatric pneumonia. This study is unique in the sense that it applied a combination of recombinant human interferon-α1b and Ambroxol on children with pneumonia, with a view to investigating its impact on inflammatory and immune indicators.

Study participants
Children with pneumonia who were admitted to Cangzhou Central Hospital from January 2019 to November 2020 were enrolled. The included children were: those who met the clinical diagnostic criteria for pneumonia, children aged more than 5 months, and who were tolerant to the study drugs, and those whose parents/guardians voluntarily participated in the study and signed informed consent. Patients who had pulmonary tuberculosis, asthma, bronchial foreign body and other respiratory diseases and pulmonary dysplasia; and those with congenital heart disease, severe liver and kidney insufficiency and sepsis were excluded. Moreover, patients with epilepsy and other central nervous system diseases, and those who used glucocorticoid within one month prior to the study were excluded.
In all, a total of 140 children were included. They were randomly assigned into control group and study group using the random number table method, 70 children each. This study was commenced following clearance from the ethical committee of our institution. The baseline data were homogenous in the two groups (p > 0.05; Table 1).
Those aged > 12 months were administered atomized inhalation of recombinant human interferon-α1b (Shenzhen Branch Xing Biological Engineering Co. Ltd; SFDA approval number S10960058; specification: 10 μg/piece) at a dose of 1.0 μg/kg + 30 mg of Ambroxol (Ruiyang Pharmaceutical Co. Ltd; SFDA approval no. H20173342; specification: 4 ml: 30 mg) + 2 mL of physiological saline. Both groups received atomized inhalation of oxygen at a flow rate of 6 L/min, 10 min at a time, twice a day. Both groups were treated for 7 days.
The protocol has been approved by the Medical Science Research Ethics Committee of Cangzhou Central Hospital, with the Approved No. of 2016-16(004) and followed the international guidelines for human studies [6].

Time taken before disappearance of clinical symptoms
Time taken before disappearance of clinical symptoms such as fever, cough, shortness of breath, wet rales, and lung X-ray shadows were recorded and compared.

Inflammation indicators
Inflammation indicators before treatment and 7 days after treatment including serum levels of amyloid A (SAA) and interleukin 6 (IL-6) were detected; 2 ml of blood was drawn intravenously on an empty stomach early in the morning, and the serum was separated within 2 hours, and frozen at -80 ℃. The IL-6 was detected using enzyme-linked immunosorbent assay (ELSA), the kit was purchased from Shanghai Jingguan Biological Products Co., Ltd., and the operation was carried out in strict accordance with the instructions. SAA was determined by immunoscattering rate turbidimetric method, the instrument was IMMACE double optical path immunoturbidity analyzer, and the kit was purchased from Bexkuman-coulter of the United States.

Immunity factors
Immunity factors before treatment, and 7 days after treatment were measured including serum immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG); The levels of serum IgA, IgG and IgM were measured by the nephelometric method using the IMMAGE 800 device (Beckman Coulter, Inc., Brea, CA, USA).

Clinical efficacy
Clinical efficacy after 7 days of treatment was classified as cured i.e. clinical symptoms disappeared, absence of lung X-ray shadow, and normal results from relevant laboratory indicators; improved i.e. clinical symptoms were mitigated, reduction in lung X-ray shadows, significantly improved laboratory indicators; or ineffective: i.e. no significant relief from the clinical symptoms, or the symptoms got even worse.

Incidence of adverse reactions
Incidence of adverse reactions post-medication was recorded and analyzed.

Statistical analysis
The SPSS 20.0 was software used for statistical evaluation. Counting data are presented as numbers and percentages [n (%)], and were compared with the X 2 test, while ordinal data were compared using rank sum test. Measurement data are expressed mean ± standard deviation (SD), and were examined with t-test for determination of differences between groups. Values of p < 0.05 were declared as significant differences.

Time taken for clinical symptoms to subside
A remarkably shorter time taken for symptoms to subside in the study group than the control group was observed (p < 0.05), as presented in Table  2.

Inflammation indices
Statistically significant reductions were seen in serum levels of SAA and IL-6 in the two groups after treatment, when compared the corresponding pre-treatment levels. However, the levels of these inflammation parameters were lower in the study group (p<0.05). These results are shown in Table 3.

Levels of immunity indices
As shown in Table 4, the levels of IgA, IgM, and IgG in the two groups after treatment were markedly elevated, but they were higher in the study group than in the control group (p < 0.05). Values are mean ± SD     Table 5 shows that clinical efficacy in the study group was markedly superior to that of the control group (p < 0.05).

Adverse events
During the treatment period, there were 2 cases of nausea, 1 case of vomiting, 1 case of rash, and 1 case of diarrhea in the study group, while in the control group, there were nausea, vomiting, and rash (one case each). None of these adverse events had a detrimental effect on the treatment, and they all disappeared on their own after the medications were stopped. No statistically significant differences in the incidence of adverse reactions were found between the two groups (p > 0.05). These data are presented in Table 6.

DISCUSSION
Serum amyloid A (SAA) arises from tissue amyloid A secreted by hepatocytes. Healthy people exhibit lower levels of SAA, while the level rises rapidly when the body is exposed to inflammation or active disease. Similar to Creactive protein, it belongs to the acute phase reactive protein. However, in contrast, under inflammatory stimulation, SAA accumulates more significantly than C-reactive protein, and the serum level of SAA rises at a much faster rate than that of C-reactive protein [7,8]. IL-6 is a proinflammatory factor secreted by Th2 cells. It stimulates a large number of phagocytes and effector molecules to gather at the site of infection to eliminate pathogens. If the antigen is not cleared in time, it will cause cytokine release levels to exceed the physiological levels, thereby leading to inflammatory infiltration [9,10]. Accumulating evidence have shown that serum SAA and IL-6 levels in children with pneumonia are elevated [11,12].
Several trials have demonstrated that pediatric pneumonia is directly linked to immune function [13,14]. Thus, the levels of serum IgM, IgG, and IgA in children with pediatric pneumonia are strikingly decreased [15]. Ambroxol is a mucolytic agent which contains the bioactive compound bromocyclohexylamine, with good bronchial and lung tissue affinity. Its mechanism of action in children with pneumonia can be explained thus: (1) stimulation of the secretions from mucous and serous glands so as to dilute sputum, promote cilia movement, and expel sputum; and (2) promotion of the synthesis of alveolar type Ⅱ cells and the release of surface-active substances to reduce the surface tension of the alveoli, inhibit contraction of smooth muscles of the respiratory tract and improve lung function, ultimately resulting in antitussive effect [16,17]. In addition, Ambroxol inhibits the growth of bacteria or viruses; this can increase the airway concentration of antibacterial drugs, and improve the anti-infective effect, thereby reducing local inflammation [18].
In this study, substantial mitigation of clinical symptoms, as well as clinical efficacy and desirable changes in related laboratory indices were observed in the two groups post-treatment. Nonetheless, the findings in this study revealed that the clinical symptoms in children treated with aerosol inhalation of combination of recombinant human interferon-α1b and Ambroxol subsided faster than those in the control group. This suggests that combined treatment using recombinant human interferon-α1b and Ambroxol further reduced the clinical symptoms of pneumonia in children. Serum SAA and IL-6 levels in the study group after treatment were found to be strikingly lower than the corresponding control group values. Thus, it can be reasonably assumed that the combination treatment with recombinant human interferon-α1b and Ambroxol plays a pivotal role in reducing the inflammation in children. Furthermore, a considerably lower serum levels of IgA, IgM, and IgG in the study group after treatment than those in the control group was noticed.
Therefore, it can be speculated that the combined treatment using recombinant human interferon-α1b and Ambroxol can effectively improve immunity in the children and promote functional immunity restoration. Moreover, an excellent clinical efficacy was seen in the study group in contrast to the control group, indicating that the combination of interferon-α1b and Ambroxol further improved the efficacy of treatment. Thus, the combined treatment option is beneficial and clinically acceptable. These aforementioned benefits may be attributed to the fact that interferon is a very special bioactive protein with multiple functions. It exerts potent and broad-spectrum antiviral effects, as well as favorable regulatory effect on immunity. Interferon a1b, one of the subtypes of interferons, has extremely potent activity, and it is directly deposited and distributed in every tissue of the respiratory system after inhalation. Therefore, the concentration of interferon is greatly increased, resulting in rapid effectiveness in removal of pathogens, inhibition of inflammation, enhancement of immunity, mitigation of clinical symptoms and suppression of the disease.
Moreover, the current study revealed that there were no serious adverse reactions during the treatment of the two groups of children, suggesting that the combination of interferon-α1b and Ambroxol is safe. Therefore, the combination of interferon-α1b and Ambroxol may be a boon in the treatment of children with pneumonia due to its innumerable benefits in terms of efficacy, inflammatory response, and serum SAA levels.

Limitations of the study
The current study is limited by a small sample size and absence of a long-term follow-up. Hence, long-term data and more participants will be needed to draw definite conclusions and use in clinical settings.

CONCLUSION
Altogether, we recommend the combination of interferon-α1b and Ambroxol as a more excellent route for the treatment of children with pneumonia, as it shows robust outcomes in producing better efficacy, yielding lower inflammatory reactions and adverse events.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

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