Effects of Probiotics Supplementation on CRP, IL-6, and Length of ICU Stay in Traumatic Brain Injuries and Multiple Trauma Patients: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Method This meta-analysis aims to evaluate the effectiveness of probiotics in reducing inflammatory biomarkers and the length of intensive care unit (ICU) stays. PubMed-Medline, SCOPUS, Embase, and Google Scholar databases up to July 2021 were searched. The meta-analysis was carried out using random-effect analysis. To determine the sources of heterogeneity, subgroup analyses were performed. In case of the presence of publication bias, trim and fill analysis was carried out. The Cochrane Collaboration tool was used for checking the quality assessment. We hypothesized that probiotics would improve inflammatory markers (CRP and IL-6) and the length of ICU stay in traumatic brain injury and multiple trauma patients. Results The present meta-analysis, which includes a total of seven studies, showed that there were no significant effects of probiotics supplementation on interleukin (IL)-6 (Hedges's g = −2.46 pg/ml; 95% CI: −12.16, 7.25; P=0.39), C-reactive protein (CRP) (Hedges's g = −1.10 mg/L; 95% CI: −2.27, 0.06; P=0.06), and the length of staying in ICU. The overall number of RCTs included in the analysis and the total sample size were insufficient to make firm conclusions. Conclusion As a result, more carefully designed RCTs are needed to investigate the effect of probiotics on inflammatory biomarkers and the length of ICU stay in traumatic brain injuries and multiple trauma patients in greater detail.


Introduction
Traumatic injuries are the leading cause of morbidity and death in young people, and the incidence of trauma in patients with serious injuries is increasing [1][2][3]. Te incidence rate of TBI is generally higher in developing nations than in more developed countries, and by 2030, it is expected to surpass several diseases as the leading cause of death and disability. For instance, the TBI incidence rate in Asia is 344 per 100,000 [4]. Nearly 80% of trauma deaths are caused by hemorrhage, central nervous system damage, or a combination of both [5]. Multiple immunological dysfunctions and metabolic changes are the subsequent life-threatening conditions of multiple traumas [6]. Immune system dysfunction is directly related to an increased risk of infection in trauma patients. Increased infectious complications in critically ill patients due to immune response impairment result in a higher mortality rate [7,8]. Traumatic brain injury (TBI) is associated with immune system dysfunction and is often the result of a collision with a motor vehicle, violence, or a fall [9][10][11]. One of the more valuable therapeutic agents to decrease illnesses triggered by oxidative stress is natural compound-based antioxidants because they play a protective role in preventing the generation of free radicals [12,13]. Recently, the importance of diet and antioxidants and complementary therapies in critically ill patients has been identifed [14]. Several clinical studies have examined the efect of antioxidant micronutrients as monotherapy or combination treatment as a component of an antioxidant strategy for critically ill systemic infammatory response syndrome (SIRS) patients [15,16]. Te results of recent studies show that in critically ill patients, the most severe cases of SIRS are associated with the most severe antioxidant reduction [17].
Probiotics are living bacteria that provide health benefts to the host when administered in sufcient amounts [18][19][20]. Prebiotics are foods or supplements that include nonliving, indigestible fbers that selectively increase the growth and/or activity of benefcial indigenous probiotic bacteria [21]. Many clinical trials and meta-analysis eforts have focused on the role of reducing ventilator-associated pneumonia. Probiotics appear to decrease infectious complications, including ventilator-associated pneumonia, and may infuence the intensive care unit mortality [22]. Recent studies on probiotic use in critical care have focused on a range of outcomes after critical illness [23]. In a study performed in 2017, the consumption of probiotic yogurt showed benefcial efects on blood glucose, glycated hemoglobin, blood pressure, and serum lipid levels [20]. Synbiotics derived from a mix of prebiotics and probiotics function synergistically to support healthy gut fora. Mixed-strain microorganisms may have a more substantial impact on the gut microbiota [24]. Several mechanisms, such as immunoglobulin A production and mucus stimulation, suppression of nuclear factor kappa B (NF-kB) activation in epithelial cells, anti-oxidative efects, liberation of antimicrobial factors, and alteration of intestinal fora by inducing antimicrobial peptides in host cells, have been proposed for the benefts of probiotics [25][26][27].
Recently, there has been a great deal of interest in the use of probiotics to decrease infammatory factors and oxidative stress markers [28]. It has been shown that probiotics suppress the expression of infammatory cytokines such as NF-kB, interleukin (IL)-6, IL-10, C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α) [29,30]. Infammation rapidly increases due to immunological disruption, which may increase the length of an intensive care unit (ICU) stay following trauma [31,32].
Te length of ICU and hospital stays was signifcantly reduced after consuming probiotics such as Lactobacillus, Bifdobacterium, and Streptococcus [33]. Based on a recent meta-analysis in 2017, probiotic administration can considerably decrease serum/plasma CRP levels [29]. Furthermore, another meta-analysis carried out in 2019 demonstrates that probiotic supplementation in critically ill patients could signifcantly reduce serum CRP levels [34]. Additionally, in 2017, a meta-analysis showed that prebiotic and synbiotic supplementation is associated with decreased serum CRP levels [35].
Several studies have demonstrated the potential efects of probiotics on infammation in diferent diseases [36][37][38]. Furthermore, several clinical trials have investigated the efects of probiotic supplementation on infammatory biomarkers and the length of stay in patients with brain trauma and multiple traumas; the results have been controversial. As a result, we conducted this study, and we hypothesized that probiotics would improve infammatory markers (CRP and IL-6) and the length of ICU stay in traumatic brain injury and multiple trauma patients. Terefore, in this systematic review and meta-analysis, we searched diferent databases for published randomized control trials (RCTs) to analyze the impact of probiotic intake on the levels of infammatory markers (CRP and IL-6) and the length of ICU stays.

Search Strategy. Te Preferred Reporting Items for
Systematic Reviews and Meta-analysis (PRISMA) statement was planned, conducted, and reported in the meta-analysis [39]. Eligible RCTs were identifed using Scopus, PubMed, Embase, and Google Scholar databases for English-language publications from the inception dates to July 2021. Supplementary Table 1 provides details of the search terms. We used a snowball sampling approach to scan the citation lists of retrieved articles for additional articles relevant to the research topics and retrieved relevant titles. Additionally, to improve the sensitivity of our search strategy, the wild-card term " * " was implied.

Study Selection Criteria, as Well as Inclusion and
Exclusion Criteria. Te following inclusion criteria were used to select trials: (1) published in the English language; (2) randomized placebo-controlled trials with a parallel or cross-over design; (3) RCTs comparing probiotics or synbiotic supplements to a placebo or no intervention group; and (4) patients with trauma and brain injuries.
Exclusion criteria included the following: (1) articles without a placebo or treatment group; (2) observational articles; (3) in vitro and in vivo studies, letters, conference abstracts, reviews, or case reports; and (4) inadequate data on the baseline or end-of-trial.

Data Extraction.
Two independent researchers (NN and MH) extracted the following data: frst author's name, year of publication, country of origin, study design, type of probiotic microbes, number of patients, age, gender, dose, and duration of supplementation. Any discrepancies were discussed and resolved with a third author (MZ).

Quality Assessment.
Te methodological quality of included studies was independently evaluated by two researchers (NN and MH) using the Cochrane risk of bias criteria: random sequence generation, allocation concealment, personnel's, assessors', and participants' blinding, incomplete outcome data, selective outcome reporting, and 2 Evidence-Based Complementary and Alternative Medicine other possible causes of bias [40]. Each quality item was stratifed as low risk, high risk, or unclear risk of bias.

Summary of Evidence: Grade Criteria.
According to the Cochrane Handbook for systematic reviews of interventions, we used the GRADE approach to evaluate the overall quality of the evidence. Each outcome was evaluated using fve criteria: (1) bias risk, (2) consistency, (3) directness, (4) precision, and (5) publication bias [41]. Four quality levels were determined using the GRADE test: high, moderate, low, and very low. Te quality declined by one level when each factor was not met [42].
2.6. Statistical Analysis. Te mean diferences and standard errors of serum/plasma CRP, IL-6, and stay in ICU between probiotic, synbiotic, and control groups were used to calculate the overall efect sizes. A standard deviation (SD) was determined using the following formula when a standard error (SE) was given instead of SD: SD � SEM × square root (n), where "n" is the number of samples per group [43]. Te random-efect model for the standardized mean diference was used in all analyses. Random-efect models consider diferent sources of uncertainties, including within-study (sampling or estimation) error and between-study variance, while fxed-efect models take only the within-study error into account. Terefore, a random-efect model can provide more conservative results than a fxed-efect model. Te efect size was estimated based on Hedges's g for staying in the ICU and serum/plasma levels of CRP and IL-6. Due to the small number of included studies, the Hartung-Knapp adjustment was performed. Te heterogeneity of included studies was assessed using Cochran's Q test and I 2 statistic [44]. Te I 2 index I 2 > 50% was deemed remarkable for heterogeneity. To investigate possible sources of heterogeneity, subgroup analysis was performed according to the duration of intervention and patients' age for the CRP factor. However, subgroup analysis was performed based on age for stays in ICU. Egger's regression test was used to assess the presence of publication bias [45]. Statistical analysis was performed using Stata software, version 16 (Stata-Corp., College Station, TX, USA). P value of less than 0.05 was recognized as statistically signifcant.

Study Selection.
Te initial search yielded 5219 results. After removing duplicates, 4066 titles and abstracts were screened for possibly relevant RCTs. Finally, seven studies were enrolled in the meta-analysis following the evaluation of these studies. Te strength of agreement using the kappa coefcient measures the agreement between two reviewers. In this study, kappa statistics were approximately 0.80 ( Figure 1). presents the articles included in this review. Te characteristics of the studies included in Table 1 were published between 2006 and 2020.

Study Characteristics.
Tere were fve studies that assessed serum/plasma levels of CRP, three for IL-6, and four studies on staying in the ICU. In the present meta-analysis, a total of 413 participants were included. Tese samples consisted of trauma patients and traumatic brain injuries. Te duration of the intervention ranged from 7 to 56 days.

Risk of Bias Assessment.
Te Cochrane collaboration's parameters were examined by two reviewers, and they reached 100% agreement. All of the studies contained suffcient information about random sequence generation.
Most of the studies reported inadequate information about allocation concealment [30,[46][47][48][49][50]. However, the majority of the studies reported adequate information about the blinding of participants and researchers and the blinding of outcome assessment [47][48][49][50][51]. Selective reporting was at a high risk of bias only in one trial [47]. Incomplete outcome data in all of the studies showed a low risk of bias. Figure 2 shows the quality of the bias assessment in detail. Figure 3(a) shows a forest plot of the pooled efect of probiotic/synbiotic consumption on plasma CRP levels. Tere were seven trials with 413 patients that compared serum/plasma CRP levels between the control and intervention groups. Probiotic/ synbiotic supplementation had no signifcant efect on plasma levels of CRP (Hedges's g � −1.10 mg/L; 95% CI: −2.27, 0.06; P � 0.06). Te between-study heterogeneity was considerable (I 2 � 90.38%, P < 0.001), that age and type of supplement were identifed as the sources of it (Table 2). Tere was a signifcant decrease in serum/plasma CRP in the subgroup of patients with a mean age of >40 years (SMD � −0.742 mg/L; 95% CI: −1.061, −0.423; P < 0.001) and duration of intervention ≥21 days (SMD � −0.722 mg/L; 95% CI: −1.166, −0.277; P � 0.001).

Efects of Probiotic/Synbiotic on CRP.
In the sensitivity analysis, the exclusion of any single study did not afect the overall estimate for the impact of probiotic/synbiotic supplementation on serum/plasma CRP concentrations (95% CI: −1.895, −0.334). Te CRP funnel plot supports the existence of a publication bias (Figure 3(b)). However, no evidence of publication bias was found using Egger's test (P � 0.672). Te overall quality of CRP evidence was moderate, which was further declined by inconsistency based on the GRADE approach (Table 3).
Removing any single study in the sensitivity analysis showed no efect on the overall estimate for the impact of probiotic/synbiotic supplementation on plasma IL-6 concentrations (95% CI: −5.682, 0.770). According to the GRADE approach, the total quality of the evidence for IL-6 Evidence-Based Complementary and Alternative Medicine was considered low because of inconsistency and imprecision (Table 3). Figure 4 indicates the forest plot of the pooled efect of probiotic/synbiotic intake on the length of stay in the ICU. Te result of four our studies which included 258 patients demonstrated that probiotic/synbiotic supplementation had no signifcant efect on the length of staying in ICU (Hedges's g � −0.29 days; 95% CI: −0.98, 0.40; P � 0.31) (Figure 5(a)).

Efects of Probiotic/Synbiotic on the Length of Staying in ICU.
Tere was evidence of signifcant between-study heterogeneity (I 2 � 78.33%, P < 0.001). Tere were no signifcant efects of probiotic/synbiotic on the length of stay in ICU after subgroup analysis by mean age.
Removing any single study in the sensitivity analysis did not afect the overall estimate for the impact of probiotic/synbiotic supplementation on the length of stay in ICU (CI: −5.453, 0.912). Furthermore, the funnel plot supports the absence of a publication bias ( Figure 5(b)). Te Egger test also confrms the absence of publication bias. Tere was no evidence of publication bias using Egger's test (P � 0.071). Te GRADE system was used to calculate the quality of evidence for the length of stay in the ICU (based on inconsistency and imprecision) ( Table 3).

Discussion
Tis study systematically reviewed seven RCTs to investigate the impacts of probiotics/synbiotics supplements on infammatory biomarkers (serum/plasma levels of CRP and  Evidence-Based Complementary and Alternative Medicine IL-6) and the length of ICU stay among trauma and traumatic brain injury patients. Te pooled analysis indicated that probiotics/synbiotics supplementation did not afect serum/plasma CRP levels signifcantly compared with control groups. Supplementation with probiotics and synbiotics had no efect on serum or plasma IL-6 concentrations or the length of ICU stays. Our hypotheses about reduced levels of CRP, IL-6, and length of ICU stays were not fully substantiated.
Tere was evidence of high heterogeneity in the data. We performed subgroup and sensitivity analyses to fnd the sources of heterogeneity. In terms of CRP and IL-6, the heterogeneity seemed to be explained by one study [30], and when this was excluded, there was no evidence of heterogeneity. It should be noted that this study was conducted on young people. Te efect of probiotics on severe infammation in younger ages might be explained by dysregulated infammatory pathways caused by age-related obesity. In the elderly, chronic low-grade infammation may impair the therapeutic efects of probiotic consumption [52].
Te stress response after trauma has been extensively studied, and it includes signifcant electrolytic, hormonal, and metabolic changes, as well as cytokine release [53,54]. Te stress reaction causes splanchnic vasoconstriction, leading to hypoxia and ischemia in the gut tissue after severe trauma [55]. Te gut microbiota regulates the immune system by producing molecules that have immunemodulatory and anti-infammatory activities and stimulate immune cells [56].
Nutritional support is a crucial issue in intensive care units, and using some compounds with anti-infammatory Kotzampassi   and antioxidant activators is thought to be benefcial for critically ill patients [16]. Probiotics can help prevent or repair "perforated" epithelial barriers as well as infuence the infammatory response indirectly by counteracting the source of the pro-infammatory stimulus associated with low endotoxemia [57]. According to recent clinical data, the administration of probiotics can decrease ICU infections [23]. CRP is a clinically important hepatic-derivedacute-phase protein that rises in response to IL-6 secretion by T cells and macrophages [58,59]. CRP levels increased in patients with multiple injuries in the early period after trauma, especially on the second day. Terefore, monitoring CRP parameters could be useful to recognize patients who are susceptible to infection in the frst two days after hospitalization [60]. Our fndings show that probiotic/synbiotic supplementation had no efect on serum/plasma CRP levels in both short and long intervention periods (i.e., <21 days and ≥21 days). However, in the mean age subgroup analysis, a reduced serum/plasma level of CRP was observed among those aged over 40 years. A chronic infammatory condition which cannot be sufciently resolved or restrained arises primarily as individuals age. On the other hand, trauma aggravates infammatory conditions, and the immune system appears to be unable to control the infammatory response punctually [61]. As a result, CRP reduction may be attributed to the elevated CRP levels and, therefore, is efective in patients aged >40 years compared with those aged <40 years.
IL-6 is a prototypical cytokine for preserving homeostasis. When homeostasis is disturbed after infection or tissue damage, IL-6 is instantly produced to assist the host's defense against such stress by stimulating acute-phase and immunological responses [62]. Te efect of probiotic/synbiotic supplementation in three studies on serum/plasma IL- Because serum/plasma CRP was signifcantly reduced in our meta-analysis and IL-6 is also the main activator of CRP [60], it appears that additional studies with a sufcient reasonable sample size should be performed to fnd serum/ plasma CRP and IL-6 associations with probiotic/synbiotic supplementation.
Due to immunological disturbance following a severe traumatic injury, it seems that patients are more vulnerable to nosocomial infections, resulting in an extended length of ICU stay [32]. Te meta-analysis carried out by Gu et al. analyzed trauma outcomes with two studies and reported that the length of ICU stay was reduced with probiotic supplementation [63]. However, despite including further studies, the length of ICU stay was not signifcantly reduced in our meta-analysis. Te Glasgow Coma Scale is a crucial measure to classify trauma severity. Te usual standard for determining whether a head injury is severe or moderate to mild is the Glasgow Coma Scale (GCS) ≤ 8 [64]. Te management of patients is commonly dependent on GCS categorization [64]. Due to the low GCS (less than 8) in some studies, probiotic supplementation may improve GCS and reduce the length of ICU stay [30,46,51].
Probiotic consumption may reduce infammatory biomarkers by increasing short-chain fatty acid (SCFA) production in the gut [65]. SCFA may quench the synthesis of hepatic CRP and result in reduced infammation and oxidative stress [66]. Probiotics also increase the production of antimicrobial peptides, which impact the mucosa's infammation resolution pathways and promote the production of several regulatory cytokines by stimulating the development and activity of immune cells such as dendritic cells and T cells [67,68]. Butyrate, as an SCFA, infuences pathways inhibiting NF-kB-induced increases in proinfammatory cytokines through some mechanisms [69]. It has also been proposed that probiotic administration reduces infammation and oxidative stress by raising glutathione levels, hydroxyl radicals, and scavenging superoxide, thereby reducing IL-6 production in adipocytes [70,71]. Furthermore, probiotics are recognized for their ability to stimulate adenosine monophosphate (AMP) production and activity. Probiotics such as lactobacillus fermentum have been shown to stimulate the production of AMP human beta-defensin-2 via pro-infammatory pathways including the transcription factors NF-kB and activator protein (AP)-1, as well as mitogen-activated protein kinase (MAPK). In addition, probiotics may be able to assist in modulating the infammatory response indirectly by increasing AMP production and secretion [72]. In addition, the binding of probiotics and microfora to innate immune system receptors such as toll-like receptors (TLRs) and Nod-like receptors (NLRs) helps regulate major intracellular pathways, which in turn helps maintain a homeostatic balance between anti-infammatory and antiinfammatory reactions at mucosal surfaces [69]. Te major strength of the present study is that it is the frst to investigate the efects of probiotic supplementation on CRP, IL-6, and the length of ICU stay in traumatic brain injuries and multiple traumas patients as a systematic review and meta-analysis of randomized controlled trials. Moreover, some limitations need to be noted. Due to the many strains and dosages of probiotics, we could not assess the efect of a specifc probiotic strain and its dosage on trauma patients. It appears that more RCTs are needed to distinguish the alteration mechanism of infammatory biomarkers by specifc probiotic strains with proper dosages. Due to fewer studies, we could not evaluate the results of the infammatory factor interleukin-6 in diferent subgroups. Furthermore, a protocol has not been preregistered for this review.

Conclusion
Te main result of this meta-analysis was that probiotic/ synbiotic supplementation did not afect serum/plasma CRP and IL-6 levels or the length of stay in the ICU. Although it should be noted that after performing the subsequent subgroup analysis of CRP based on age and intervention duration, it reveals that probiotic/synbiotic supplementation in the >45-yearold subgroup and both durations (a subgroup of >21 and ≤21 days) showed benefcial impacts. More comprehensive, well-designed clinical trial studies with appropriate sample sizes are needed to determine the efect of probiotic/synbiotic on IL-6 and the length of stay in the ICU in patients with traumatic brain injuries and multiple traumas.

AMP:
Adenosine monophosphate CRP: C-reactive protein GCS: Glasgow coma scale IgA: Immunoglobulin ICU: Intensive care unit IL-6: Interleukin MAPK: Mitogen-activated protein kinase NLRs: Nod-like receptors NF-kB: Nuclear factor kappa B PRISMA: Preferred reporting items for systematic reviews and meta-analysis RCT: Randomized control trials SCFA: Short-chain fatty acids Funnel plot with pseudo 95% confidence limits Standard deviation SE: Standard error SMD: Standard mean diference TLRs: Toll-like receptors TNF-a: Tumor necrosis factor-alpha WMD: Weighted mean diference SIRS: Systemic infammatory response syndrome.

Conflicts of Interest
All authors declare that there are no conficts of interest.