Effects of vitamin D on apoptosis of T-lymphocyte subsets in neonatal sepsis
- Authors:
- Published online on: May 24, 2018 https://doi.org/10.3892/etm.2018.6215
- Pages: 629-634
Abstract
Introduction
Neonatal sepsis is one of the most common critical diseases in pediatrics, with a higher incidence, which can be caused by a variety of pathogenic bacteria infections and often accompanied by systemic inflammatory response syndrome (SIRS). In the neonatal period, especially in premature infants, when infection occurs, blood circulation is invaded by bacteria that reproduces and produces toxins, resulting in systemic reactions (1,2). Because of insidious onset, there are no specific clinical symptoms in child patients during early stage; given that the rapid progress of the disease will lead to secondary organ damage, there is a need for early treatment, which is a huge difficulty and challenge in clinical practice; the lack of early treatment easily leads to death of children (3,4). Vitamin D is an essential nutrient; vitamin D deficiency can easily cause rickets and chondropathy. In recent years, studies by Chiesa et al (5), El-Mazary et al (6) and Cetinkaya et al (7) have shown that vitamin D plays an important role in immunoregulation. In this study, 150 neonates with sepsis and 100 healthy newborns were selected, and the levels of T-lymphocyte subsets, tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and calcitonin (CT) were detected, so as to investigate the influence of vitamin D on apoptosis of T-lymphocyte subsets, the effect on inflammatory factors and its possible mechanism.
Patients and methods
Objects of study
One hundred and fifty neonatal patients with sepsis admitted to Ruian People's Hospital (Wenzhou, China) from October 2015 to May 2017 were selected and randomly divided into observation group (n=75) and treatment control group (n=75), while 100 healthy newborns during the same period were enrolled as healthy control group. All the patients in the observation group and the treatment control group had positive blood culture results, and the imaging findings were in accordance with the positive standard diagnosis. Inclusion criteria: Observation group and treatment control group, patients aged <28 days; patients with sepsis. Exclusion criteria: Patients with immune dysfunction; patients with immune system disease; children with incomplete data; children had administration with vitamin D; patients with liver or kidney dysfunction; patients who gave up treatment. Normal control group: Neonatal without sepsis.
The parents of the patients or their family members signed the informed consent, and this study was approved by the Ethics Committee of Ruian People's Hospital (Wenzhou, China).
Treatment methods
We initially collected 333 patients with neonatal sepsis from October 2015 to May 2017 in Ruian People's Hospital, while 183 patients were excluded according to exclusion criteria. Therefore, only 150 patients with neonatal sepsis were included. They were randomly divided into observation (n=75) and treatment control group (n=75). Both treatment control and observation group received fluid resuscitation and routine anti-infection treatment. Based on it, the observation group was treated by intramuscular injection with 0.25 µg/per child vitamin D (8,9); when the condition was improved and remained stable, the dosage of medication could be reduced until withdrawal (Fig. 1).
Detection methods and evaluation
Observation indicators: Cluster of differentiation 8 (CD8+) T-cells, CD4+ T-cells, TNF-α, IL-1 and CT before treatment and at 72 h after treatment. T-lymphocyte subsets, CD8+ T-cells and CD4+ T-cells were detected by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). TNF-α, IL-1 and CT were determined by enzyme-linked immunosorbent assay (ELISA), and the kits were provided by Jingmei Biotech Co., Ltd. (Shanghai, China). Serum 25(OH)D was detected by Roche Diagnostics GmbH (Mannheim, Germany) Cobas e411 automatic electrochemiluminescence immunoassay analyzer. Baseline levels were detected in the healthy control group.
Statistical analysis
Data were processed SPSS 22.0 (IBM Corp., Armonk, NY, USA). Data conforming to normal distribution are expressed as mean ± standard deviation and compared by t-test, and nonparametric rank sum test was used for those meeting abnormal distribution. The Chi-square test was adopted for the comparison of rate. P<0.05 was considered to indicate a statistically significant difference.
Results
Clinical data of the three groups
There were 75 patients in the observation group, including 39 males and 36 females, aged 1–10 days, with an average of 4.71±1.59 days; there were 75 cases in the treatment control group, including 37 males and 38 females, aged 1–10 days, with an average of 4.45±1.42 days; there were 100 patients in the healthy control group, including 50 males and 50 females, aged 1–10 days, with an average of 4.67±1.62 days. The sex, age, mode of birth and other general data of the three groups were comparable, the differences were not statistically significant (P>0.05) (Table I).
Test results of vitamin D in each group
There were statistically significant differences in comparisons of 25(OH)D among three groups. Serum 25(OH)D level in the observation group was higher than that in the treatment control group (P<0.05), but the levels in the two groups were remarkably lower than that in the healthy control group, and the differences were statistically significant (P<0.05) (Table II).
Comparison of T-lymphocyte subsets, inflammatory factor and CT in three groups before and after treatment
The difference in comparison of CD4+ T-cells between sepsis patients and healthy control group had statistical significance before treatment (P<0.01), and it showed a statistically significant difference after treatment with vitamin D for 72 h between the observation group and the treatment control group (P<0.05). The difference in comparison of CD8+ T-cells between sepsis patients and healthy people had no statistical significance before treatment (P>0.05), but it showed a statistically significant difference after treatment for 72 h between the observation group and the treatment control group (P<0.01). After treatment for 72 h, CD4+ T-cells were increased, and the ratio of CD4+ to CD8+ was close to 1. The differences in comparisons of inflammatory factor levels between children with sepsis and healthy control group were statistically significant before treatment (P<0.01). The differences in comparisons of TNF-α, IL-1 and CT levels between the observation group and the treatment control group were statistically significant after treatment with vitamin D for 72 h (P<0.05) (Figs. 2 and 3).
Logistic regression analysis of vitamin D, CD4+ T-lymphocytes and CD8+ T-lymphocytes
There was a positive correlation between vitamin D and CD4+ T-lymphocytes and CD8+ T-lymphocytes (r=0.712, P=0.001; r=0.685, P=0.003) (Fig. 4).
Cox regression analysis
We analyzed the possibility of vitamin D, CD4+ T-lymphocytes and CD8+ T-lymphocytes as the risk factors for sepsis. The median vitamin D level before treatment was 15.82 mg/l and the median percentage of CD4+ T-lymphocytes was 26.58%, and the median percentage of CD8+ T-lymphocytes was 21.75%. Patients were divided into high and low levels according to the median level of CD8+ T-lymphocytes. The results showed that they may be risk factors for sepsis (Table III).
The outcomes of patients in different groups
The mortality rate of sepsis children with vitamin D level <15.82 mg/l is 35.2%. The mortality rate of sepsis children with vitamin D level >15.82 mg/l is 11.3%. The difference is statistically significant (P<0.05). The AUC of the ROC curve analysis of vitamin D levels predicted the prognosis of children with sepsis was 0.743, which had a good predictive value (P<0.05) (Fig. 5).
Discussion
Neonatal sepsis is a common clinical syndrome in newborns, with high morbidity and mortality. As the disease is relatively insidious, the condition is usually in a state of danger when it is found, involving complex immune dysfunction, abnormal coagulation and other aspects; systemic immune response easily occurs under the induction of infection in neonates with deficiencies in immune function (10). Admittedly, the disease can be controlled to a certain extent by adopting antibiotics, but at the same time, the bacteria died from antibiotics can release large amounts of endotoxin, resulting in the rapid increase in endotoxin levels in patients, thus activating immune response; on the contrary, it will aggravate the disease (11). The studies by Yang et al (12) and Cizmeci et al (13) have revealed that vitamin D has an important regulatory role in the immune system, which can inhibit cell proliferation and maturation, and regulate inflammatory cytokines. Xiao et al (14) and Nzegwu et al (15) investigated the relationship between vitamin D and sepsis. By detecting apoptosis of T-lymphocyte subsets and inflammatory factors, this study aimed to investigate the effect of vitamin D on apoptosis of T-lymphocyte subsets in neonatal sepsis.
Compared to other studies, the objects of this study were selected in strict accordance with inclusion and exclusion criteria, ensuring the reliability of the study. At present, there is little research on the immunological regulation mechanism of vitamin D on neonatal sepsis; however, this study investigated the possible immune regulation mechanism of vitamin D on neonatal sepsis, displaying better innovation.
As known, T-lymphocytes mainly include CD4+ T-cells, CD8+ T-cells, which play important roles in the organism (16,17). Moreover, the size of CD4+ T-cells is closely related to immunosuppression (18). In this study, the difference in comparison of CD4+ T-cells was statistically significant after treatment with vitamin D for 72 h betweent the observation group and the treatment control group. After treatment with vitamin D for 72 h, the amounts of CD4+ T-cells and CD8+ T-cells were increased, and the increase of CD4+ T-cells was more remarkable. We analyzed the correlation between vitamin D and CD4+ T-lymphocytes and CD8+ T-lymphatic level. The results of logistics regression analysis showed that the correlation coefficient was 0.712, which showed that vitamin D may be able to regulate the levels of CD4+ T-lymphocyte and CD8+ T-lymphocytes. Chen et al (19) found that vitamin D can regulate the proliferation of CD8+ T cells in patients with gastroenteritis and promote the development of gastrointestinal inflammation. Cantorna et al (20) also found that vitamin D can regulate T-lymphocyte levels in mouse. Therefore, we sepeculate that vitamin D is associated with the prognosis of neonatal sepsis. Thus, the role of vitamin D in neonatal sepsis may be achieved by reducing apoptosis in T cells via inhibiting excessive immune response (21). Under the action of inflammatory factors, nuclear factor (NF-κB) is activated, which induces inflammatory cell infiltration, thus leading to immune dysfunction (22,23). We will further test this hypothesis through in vitro experiment in our future studies. This study indicated that the differences in comparison of TNF-α, IL-1 and CT levels between the observation group and the treatment control group were statistically significant before treatment, and after treatment with vitamin D for 72 h, the levels of these indicators in the observation group were lower than those in the treatment control group. The high levels of inflammatory factors were obviously ameliorated, which reduced damage to the body to a certain extent. We also analyzed the prognosis of children with sepsis. We divided the children into low level group (≥15.82 mg/l) and high level group (<15.82 mg/l) according to the level of vitamin D. There is significant difference in mortality rate between two groups of children. The AUC was 0.743, indicating that vitamin D may have good prognostic value in children with sepsis.
In this study, given that there may be some bias because of the limited sample size, and the inappropriate dose of vitamin D may cause hypercalcemia (24), the dosage, time and mode of medication and other specific issues still need to be further investigated by experts worldwide. Vitamin D may be a risk factor for sepsis, but more research and larger samples are needed to verify whether it is an independent factor. More studies are needed to investigate the applications value of vitamin D in the diagnosis of sepsis. It is hoped that individualized treatment of neonatal sepsis can be carried out from the perspective of combined treatment, so as to reduce the morbidity and mortality of neonatal sepsis in China.
In conclusion, vitamin D can enhance the immune function of neonates with sepsis by regulating the levels of inflammatory factors and T-lymphocyte subsets.
Acknowledgements
Not applicable.
Funding
This study was supported by Wenzhou Science and Technology Bureau Project (Y20150128), Wenzhou Health Bureau Project (2015B37) and Ruian Science &Technology Bureau Project (MS2017006).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
GZ and WJ conceived and designed the study. GZ, MP and ZL were responsible for the collection and analysis of the data. WX and WJ interpreted the data and drafted the manuscript. GZ revised the manuscript critically for important intellectual content. All authors read and approved the final study.
Ethics approval and consent to participate
The study was approved by the Ethics Committee of Ruian People's Hospital (Wenzhou, China). Signed informed consents were obtained from the parents of the child patients.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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