Effects of probiotics combined with early enteral nutrition on endothelin-1 and C-reactive protein levels and prognosis in patients with severe traumatic brain injury.

OBJECTIVE: To investigate the effects of probiotics combined with early enteral nutrition on levels of endothelin-1 (ET-1), C-reactive protein (CRP), and inflammatory factors, and on the prognosis of patients with severe traumatic brain injury (TBI).
METHODS: We enrolled 76 adults with severe TBI. The patients were divided randomly into two equal groups administered enteral nutrition with and without probiotics, respectively. Demographic and clinical data including age, sex, Glasgow Coma Scale score, Sequential Organ Failure Score, Acute Physiology, Chronic Health Score, hospitalization, mortality, and infections were recorded.
RESULTS: Serum levels of inflammatory factors gradually decreased with increasing treatment time in both groups. However, ET-1 at 15 days, and interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and CRP at 7 and 15 days decreased significantly more in the combined treatment group. Hospitalization duration and pulmonary infection rates were also significantly reduced in the combined compared with the enteral nutrition alone group. GCS scores at 15 days were significantly lower in the combined compared with the enteral nutrition group.
CONCLUSION: Probiotics combined with early enteral nutrition could reduce serum levels of ET-1, CRP, and IL-6, IL-10, and TNF-α, and could thus improve the recovery of patients with severe TBI.

RCT Entities:   Population Interventions Outcomes

Introduction

Traumatic brain injury (TBI) is a major health problem worldwide and one of the most common causes of death and disability in people ≤40 years old, often as a result of motor vehicle collisions, falls, or violence.[1,2] Severe TBI, defined as head trauma associated with a Glasgow Coma Scale (GCS) score of 3 to 8, is associated with a weighted average mortality of 39%, while the mortality for cases with an unfavorable outcome on the Glasgow Outcome Scale is 60%.[3] The management of severe TBI remains a clinical challenge due to its severe symptoms and poor prognosis.

Given the role of the brain as the central functional regulator in the human body, TBI may be associated with complex metabolic alterations, including aberrant cellular metabolism, hormonal changes, and inflammatory cascade.[4,5] Brain injury may also cause immunological dysfunctions, leading to hyperinflammation and abnormal expression of inflammatory factors such as interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, endothelin (ET)-1, and C-reactive protein (CRP), which may in turn contribute to secondary injuries.[6,7]

Nutritional support is important and is widely used in the intensive care of critically ill patients.[8] The effects of nutritional support have been reported in several studies, and it is considered as an important adjunctive therapy for TBI-induced metabolic disorders.[8,9] However, the importance of nutritional support is often neglected and underestimated during the clinical management of patients with TBI.[10]

The role of probiotics in the management of various infectious diseases has recently been highlighted due to their ability to restore the nonpathogenic digestive flora that are commonly lost during these conditions.[11] Probiotics are considered to modulate local and systemic immune functions, which may further improve gut mucosal barrier functions, and reduce the growth of potentially pathogenic microorganisms.[12] However, although several studies have shown that probiotics can influence serum levels of Th1/Th2 cytokines in patients with severe TBI,[13] the effect of probiotics on CRP and ET-1 in patients with severe TBI remains unknown.

In the present study, we investigated the effects of probiotics combined with early enteral nutrition on serum levels of CRP, ET-1, and other inflammatory factors, and on the prognosis of patients with severe TBI. These results could improve our understanding of the role of probiotics in TBI and provide clinical evidence for the application of probiotics in the treatment of patients with severe TBI.

Materials and methods

Patients and treatment

This prospective study enrolled consecutive adult patients with severe TBI who were treated at Shengli Oilfield Central Hospital, Dongying, China, between July 2016 and August 2017. The diagnosis of TBI was confirmed by imaging methods including computed tomography and magnetic resonance imaging. All TBIs were diagnosed as closed craniocerebral injuries. All patients were treated within 24 hours after TBI and were diagnosed with severe TBI by both clinical and iconographic methods, with a GCS score of 3 to 8. Patients with other severe infections, gastrointestinal injury or disease, and other severe diseases such as cardiac, liver, or brain diseases were excluded from the study. All the patients were divided randomly into two groups: a combined group administered enteral nutrition and probiotics, and an enteral nutrition alone group. General treatment for severe TBI was administered according to locally agreed management protocols for head injury. Written informed consent was obtained from all participants or their families within 24 hours of admission. The present study was approved by the Ethics Committee of Shengli Oilfield Central Hospital.

Patients in the combined group received both enteral nutrition and probiotics within 48 hours after admission. Enteral nutrition was administered according to the protocols of the American Society for Parenteral and Enteral Nutrition (ASPEN).[14] NengQuanLi enteral nutrition suspension (Nutricia (Wuxi) Co., Ltd., Wuxi, China) was administered at 30 kcal/kg/day (Table S1) through a nasogastric uniform pump. According to ASPEN’s recommendations, the nutrition should provide >50% to 60% of the target calories during the first week of admission, as well as accounting for the hypermetabolism and hypercatabolism in patients with severe TBI.[15] About 400 to 500 mL of nutrition suspension was given on the first day, increasing by 400 to 500 mL every day to a total of 1600 to 2000 mL. The initial administration rate was 25 mL/hour, and this generally increased to 80 to 100 mL/hour after 3 to 5 days. Probiotic treatment was administered by probiotics tablets (210 mg/per tablet) combining Bifidobacterium, Lactobacillus, and Enterococcus faecalis (Xinyi Pharmaceutical Factory Co., Ltd., Shanghai, China). The main bacteria in the tablets were Bifidobacterium longum, Lactobacillus bulgaricus, and E. faecalis ≥1.0 × 107 colony-forming units. Patients were given six tablets twice a day by gastric tube injection, or by oral administration if possible. Both treatments were continued for 15 days.

Data collection

Demographic data including age and sex, and clinical variables including type of TBI, GCS score, Sequential Organ Failure Score (SOFA), Acute Physiology, Chronic Health Score (APACHE II), hospitalization, mortality, and infections were recorded. Peripheral blood samples were collected from the elbow vein at admission (0 days), and 1, 3, 5, 7, and 15 days after admission. Serum levels of IL-6, IL-10, TNF-α, ET-1, and CRP were determined by enzyme-linked immunosorbent assay (ELISA) using commercial ELISA kits (Abcam, Cambridge, MA, USA) according to manufacturer’s instructions.

Statistical analysis

Numerical data were compared by χ2 tests and continuous data were compared by Student’s t-tests. Measured data were expressed as mean ± standard deviation. A value of P<0.05 was considered significant. All calculations were made using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA).

Results

Clinical information

Seventy-six patients with severe TBI were enrolled, including 36 men and 40 women (mean age 37.47 ± 12.24 years). Their demographic and clinical information is shown in Table 1. Patients were divided equally into a combined and an enteral nutrition group (n = 38 each). There was no significant difference in age, sex, injury type, or GCS, SOFA, or APACHE II scores between the two groups.

Table 1.

Clinical information for all patients.

Variable	Combined group (n = 38)	Enteral nutrition group (n = 38)
Age, years	35.97 ± 13.12	38.65 ± 11.26
Sex, male : female	19 : 19	17 : 21
Injury type, n (%)
Contusion	17 (44.74)	16 (42.11)
Fracture	10 (26.31)	8 (21.05)
Epidural hematoma	8 (21.05)	11 (28.95)
Subdural hematoma	10 (26.31)	7 (18.42)
Intracranial hemorrhage	3 (7.89)	3 (7.89)
Subarachnoid hemorrhage	4 (10.53)	3 (7.89)
GCS score	5.47 ± 1.59	5.79 ± 1.74
SOFA score	5.29 ± 1.35	5.02 ± 1.28
APACHE II score	13.26 ± 2.31	12.84 ± 2.37

GCS, Glasgow Coma Scale; SOFA, sequential organ failure score; APACHE II, chronic health score.

Probiotics plus early enteral nutrition decreased serum levels of inflammatory factors in patients with severe TBI

We determined the effects of enteral nutrition with and without probiotics on serum levels of inflammatory factors including IL-6, IL-10, TNF-α, ET-1, and CRP. Serum levels of all the inflammatory factors gradually decreased with increasing treatment time in both groups. However, the decreases in ET-1 at 15 days, and in IL-6, IL-10, TNF-α, and CRP at 7 and 15 days were significantly greater in the combined compared with the enteral nutrition alone group (P < 0.05) (Figure 1). These results indicated that probiotics combined with early enteral nutrition helped to reduce serum levels of ET-1, CRP, and other inflammatory factors in patients with severe TBI.

Figure 1.

Serum levels of IL-6, IL-10, TNF-α, ET-1, and CRP in patients with TBI at the indicated times after the start of nutritional support, as determined by ELISA. *P < 0.05, **P < 0.01 between the combined and enteral nutrition groups. d, days; IL, interleukin; TNF, tumor necrosis factor; ET-1, endothelin-1; CRP, C-reactive protein.

Probiotics combined with early enteral nutrition facilitated recovery in patients with severe TBI

We also analyzed the effects of enteral nutrition with and without probiotics on the clinical outcomes of patients with severe TBI. The duration of hospitalization and incidence of pulmonary infection were both significantly lower in the combined compared with the enteral nutrition group (P < 0.05) (Table 2). However, there was no significant difference in 1-month mortality rates, intracranial, incision, or blood infection rates, sepsis, septic shock, or systemic inflammatory response syndrome between the two groups. GCS scores were significantly lower in the combined group at 15 days (P < 0.05), but there was no significant difference in SOFA or APACHE II scores (Table 3). These results suggested that probiotics combined with early enteral nutrition might improve the prognosis of patients with TBI by accelerating their recovery, but had no effect on mortality.

Table 2.

Hospitalization, mortality, and infection status in all patients.

Variable	Combined group (n=38)	Enteral nutrition group (n=38)	P value
Hospitalization in ICU, days	10.32 ± 5.31	14.24 ± 6.79	<0.001
1-month mortality, n (%)	5 (13)	7 (18)	0.329
Infections, n (%)
Pulmonary infection	17 (45)	28 (74)	<0.001
Intracranial infection	5 (13)	5 (13)	>0.95
Incision infection	3 (8)	2 (5)	0.390
Blood infection	4 (11)	3 (8)	0.469
Sepsis	4 (11)	3 (8)	0.469
Septic shock	3 (8)	2 (5)	0.390
Systemic inflammatory response syndrome	3 (8)	3 (8)	>0.95

P values calculated by Students t-test for continuous data and χ2 test for rates.

Table 3.

GCS, SOFA, and APACHE II scores in patients according to nutritional support.

Group	0 days	1 day	3 days	5 days	7 days	15 days
GCS
Combined group (n = 38)	5.47 ± 1.59	6.42 ± 1.67	7.37 ± 1.76	8.45 ± 1.93	10.05 ± 2.10	12.10 ± 2.02
Enteral nutrition  group (n = 38)	5.79 ± 1.74	6.71 ± 1.86	7.55 ± 2.08	8.34 ± 2.03	9.63 ± 2.06	10.16 ± 1.42
P value	0.412	0.477	0.678	0.817	0.381	<0.001
SOFA
Combined group (n = 38)	5.29 ± 1.35	4.89 ± 1.47	4.39 ± 1.52	4.34 ± 1.53	2.94 ± 1.58	2.39 ± 1.52
Enteral nutrition  group (n = 38)	5.02 ± 1.28	4.50 ± 1.47	4.10 ± 1.43	4.05 ± 1.49	2.81 ± 1.64	2.02 ± 1.69
P value	0.387	0.244	0.395	0.414	0.723	0.337
APACHE II
Combined group (n = 38)	13.26 ± 2.31	14.60 ± 1.48	12.84 ± 1.46	10.97 ± 1.49	9.13 ± 1.73	7.21 ± 1.85
Enteral nutrition  group (n = 38)	12.84 ± 2.37	14.28 ± 1.27	12.53 ± 1.37	10.74 ± 1.62	8.87 ± 1.89	7.00 ± 2.19
P value	0.423	0.322	0.334	0.510	0.528	0.652

GCS, Glasgow Coma Scale; SOFA, sequential organ failure score; APACHE II, chronic health score. P values calculated by Student’s t-test for continuous data and χ2 test for rates.

Discussion

Metabolic disorders induced by TBI are a major problem affecting the initial period of hospitalization and stabilization, and also negatively impacting on rehabilitative treatments.[16] Both nutritional support and probiotics have been reported to be effective in the management of many diseases, including in critically ill patients in intensive care. However, studies focusing on the effect of probiotics combined with enteral nutrition on CRP and ET-1 in patients with severe TBI are lacking. In the present study, we provided the first evidence showing that probiotics combined with early enteral nutrition could reduce serum levels of CRP, ET-1, and other inflammatory factors, and could improve the clinical prognosis of patients with severe TBI.

Numerous studies have found abnormal expression of inflammatory factors in patients with TBI, and this has been considered to be a major factor contributing to their poor prognosis. Yang et al.[17] showed that IL-6 was a potential mediator of mild TBI-induced pathology and that systemic IL-6 modulated the degree of neuroinflammation and contributed to reduced motor coordination after mild TBI. Timmerman et al.[18] found that IL-6 and IL-10 expression levels were up-regulated in patients with TBI, and that patients recovering from TBI had blunted IL-6, IL-10, and cortisol responses following a peak exercise test. Su et al.[19] also showed that elevated CRP protein levels might be a prognostic predictor in patients with mild TBI. Rey et al.[20] recently found that carboxy-terminal pro-endothelin-1 was associated with increased prediction of mortality risk scores for pediatric patients in intensive care units. All these studies were consistent with our current findings. The present study also found that probiotics combined with early enteral nutrition significantly reduced serum levels of CRP and ET-1. These decreased levels of inflammatory factors might contribute to lower pulmonary infection rates, which could otherwise be aggravated by activation of these inflammatory cytokines.

Although the efficiency of probiotics remains controversial,[21] both early enteral nutrition and probiotics have been used in the treatment of TBI. Costello et al.[22] reviewed 10 years’ worth of literature regarding the nutritional treatment of TBI and found that proper nutrition could benefit patients with TB, though the optimal treatment method remains to be determined. Chapple et al.[23] found that both energy and protein levels were deficient throughout hospitalization in patients admitted with TBI, and nutritional support was therefore necessary. In a recent review, Curtis et al.[24] discussed the efficiency of nutritional treatment in patients with chronic TBI and concluded that, although nutritional treatment might contribute to lower mortality and infection rates in patients with TBI, more clinical evidence was needed to confirm the results.

Several studies have also examined the effects of probiotics on inflammatory factors. Tan et al.[13] demonstrated that probiotics decreased serum levels of IL-4, IL-6, and IL-10 and showed that treatment with probiotics might shorten the ICU stay in patients with TBI. In a recent meta-analysis, Milajerdi et al.[25] analyzed 42 clinical trials and concluded that probiotics could reduce serum levels of pro-inflammatory cytokines including, high sensitivity-CRP, TNF-α, IL-6, IL-12, and IL-4. Probiotics might prevent inflammation by inhibiting pathogenic enteric bacteria, improving epithelial and mucosal barrier functions, or altering immunoregulation.[26] In addition, the current study showed that combined treatment with enteral nutrition and probiotics could decrease GCS scores, and reduce pulmonary infection rates and hospitalization times in patients with TBI, suggesting that this combination might accelerate patient recovery. However, further studies are needed to confirm these results.

The present study also had some limitations. First, the sample size was small and all the cases were from a single center. Second, we did not investigate the molecular mechanisms responsible for the beneficial effects of probiotics, and further studies using animal models are required.

In conclusion, we conducted a prospective study to investigate the effects of probiotics combined with early enteral nutrition on serum levels of ET-1 and CRP, and on the prognosis of patients with severe TBI. The combination of probiotics with early enteral nutrition reduced serum levels of inflammatory factors and improved the clinical prognosis of patients with severe TBI. These results improve our understanding of the role of probiotics in TBI, and provide more clinical evidence for the application of probiotics in the treatment of patients with severe TBI.