The Effects of Probiotic/Synbiotic on Serum Level of Zonulin as a Biomarker of Intestinal Permeability: A Systematic Review and Meta-Analysis

Background: This systematic review and meta-analysis was conducted to obtain a conclusive result on the influence of probiotics/synbiotic on serum levels of zonulin. Data related to serum levels of zonulin were extracted to determine the effects of probiotic/synbiotic on intestinal permeability. Methods: The literature search was conducted across the Cochrane Central Register of Controlled Trials, Pub-Med, Scopus and ISI Web of Science, Search up to Nov 2018. Clinical trials evaluating the effect of probiotic/synbiotic on serum zonulin levels of all human subjects were included. Results: Nine studies (including 496 intervention and 443 control subjects) met the inclusion criteria for the meta-analysis. According to the meta-analysis, probiotic/synbiotic has a significant effect on serum zonulin reduction (WMD=−10.55 [95% CI: −17.76, −3.34]; P=0.004). However, the high level of heterogeneity was observed among the studies (I2=97.8, P<0.001). The subgroup analysis suggested study quality, blinding, study duration, Participants age, subject’s health status and supplement type as sources of heterogeneity. Conclusion: Probiotic/synbiotic have favorable effects on serum levels of zonulin as a measure of intestinal permeability. However, the results should be interpreted with caution due to the high heterogeneity and further evidence is required before definitive recommendations can be made.


Introduction
The gastrointestinal epithelium, covered by a single layer of epithelial cells, forms a protective barrier between apical and basolateral compartments. The function of the epithelial barrier mostly relies on intercellular junctions, known as tight junctions which consist of cytoplasmic plaque proteins including zonula occludens (1). One of the modulators of these tight junctions is Zonula occludens toxin (Zot) which temporarily increases the paracellular permeability without damage to the epithelium (2). Zonulin, a 47-kDa protein, is an endogenous analog of Zot that can similarly improve paracellular transport to Zot (3). Zonulin represents a physiological defensive mechanism against microorganism colonization of the small intestine (4). Altered intestinal per-meability in several pathological conditions including autoimmune diseases, diseases of the nervous systems, and neoplastic conditions has been associated with overexpression of zonulin in the intestinal mucosa (5). Moreover, antigen presentation in human macrophages appears to be regulated by zonulin. This would change the cytokine profile and subsequently potentiate the switch from immune tolerance to autoimmunity (6). Strategies aimed at modifying the intestinal barrier function through downregulating zonulin pathway suggest a potential therapeutic target for the treatment of these chronic diseases. Zonulin inhibitor Larazotide acetate featured an upcoming result in celiac disease; however, safety and efficacy of Larazotide need to be determined by large clinical trials (7). Besides conventional treatments, several nutritional compounds including Colostrum bovinum (8), Apple-Derived Pectin (9), vitamins A and D (10) have been found to modulate the epithelial barrier by reducing serum levels of zonulin. Probiotic (live microorganisms) and synbiotic (containing probiotic strains and prebiotics including inulin, starch, and fructooligosaccharides) are promising groups of nutraceuticals that affect intestinal permeability through alterations in expression of tight junction proteins (11). Several studies have been conducted to investigate the impacts of probiotics on serum or fecal levels of zonulin; however, the results were inconsistent and inconclusive (11,12). Therefore, we conducted this systematic review and meta-analysis to review systematically the influence of probiotics or synbiotic on serum levels of zonulin.

Study Selection
After that the relevance of a study was confirmed, publication's full texts were reviewed and those that fulfilled the eligibility criteria were included. The following eligibility criteria were applied: 1) published in English or Persian; 2) using probiotic/synbiotic as the supplement; 3) reporting serum zonulin as the outcome of the study. Following studies were excluded: 1) articles without full-text availability, non-English, ecological study, qualitative study, opinion pieces, conference abstracts, review articles and editorials; 2) reporting unrelated data.

Data Extraction
The data were extracted independently by 2 reviewers (MA and AR), and in the event of disagreement, a decision being made after MS cross-examined doubtful data. Studies characteristics including first author's name, publication year, country, study design, quality score, sample size, supplement and placebo composition, study duration, participant's gender, age, and health status were extracted. Moreover, to evaluate the effect of probiotic/synbiotic on zonulin concentration, the mean ± SD of the serum zonulin before and after supplementation was extracted from eligible studies.

Assessment of risk of bias
The quality of the studies was evaluated by 2 separate reviewers according to the Jadad score (13). The Jadad score considers randomization, blinding, description of withdrawals and dropouts. Each study was scored between 0-5; higher numbers represent better quality.

Statistical analysis
The effect size, estimated as the weighted mean difference (WMD), was used to perform the fixed method meta-analysis. In case of significant heterogeneity between studies, a random-effects meta-analysis was carried out (14). Heterogeneity was evaluated using the I 2 index and Cochrane's Q test. Heterogeneity was considered low if I 2 < 30%, moderate if I 2 = 30%-75%, and high if I 2 >75% (15). Subgroup analyses were performed according to study quality (low or high), blinding (yes or no), age (under 45 yr or more than 45 yr), supplement type (probiotic or synbiotic), study duration (less than 3 months or more than 3 months), and participant's health status (healthy or not healthy) to identify the potential sources of heterogeneity. In addition, sensitivity analysis and meta-regression were performed to investigate further on heterogeneity sources. Begg's rank correlation, Egger's linear regression, and funnel plots were used to examine for the presence of publication bias. All analyses were carried out using Stata, ver. 14 SE (Stata Crop, College Station, TX, USA). P-values <0.05 were considered statistically significant.

Characteristics of the studies
As shown in Fig. 1, the early electronic search resulted in 538 studies, after duplicate removal. Following the title and abstract screen, 492 studies were excluded due to reporting unrelated data, being review articles, and not be written in English.

Findings from the meta-analysis
Overall, 9 studies provided 13 effect sizes, including 496 intervention and 443 control subjects, regarding the effect of probiotic/synbiotic supplementation on the serum levels of zonulin.

Discussion
Findings of the current meta-analysis suggest that compared to placebo, supplementation with probiotic/synbiotic significantly reduces serum levels of zonulin; however, there was high heterogeneity among selected studies. When the analysis was separately performed for probiotic and synbiotic, a significant reduction was observed in those that received probiotic only. Moreover, subgroup analysis based on study quality, blinding, duration of intervention, age, and health status could explain the source of heterogeneity among studies. Zonulin is considered as a physiological modulator of intestinal permeability and a surrogate marker of the dysfunctional gut barrier (3,5,25). Certain gut microbes, in particular pathogens, might induce the release of zonulin from the gut suggesting a mechanistic link between alterations in the gut microbiota and gut barrier function (4). Despite the small sample size, our finding is of importance due to the increased levels of circulating zonulin in a wide range of chronic diseases (26)(27)(28). Although we observed heterogeneity among studies, the results were not significant when the analysis was confined to high-quality studies, blinded trials, studies with longer duration, and those recruited healthy subjects younger than 45 yr old. The supplemented probiotics may surpass certain lactic acid bacteria that activate Toll-like receptor 2 (TLR2) signaling pathway (12). TLR2 is localized in cell membranes of the intestinal epithelium. It induces epithelial resistance in the activated form (29,30). Moreover, protective role of probiotics on intestinal permeability may be explained in part by inhibiting p38 MAPK, a Ser/Thr kinase related to the upregulation of several inflammatory indices (23). In vitro studies assumed that the combination of probiotics with prebiotics might change intestinal permeability simultaneously (31,32). Therefore, we confined the analysis to supplemental synbiotic and observed that the results were no longer significant. Because of varying study populations and probiotic products, more powerful clinical trials with longer follow-up are needed to confirm this finding. Sensitivity analysis indicated that results might have been disproportionally influenced by a study (24). After excluding this trial, the meta-analysis no longer produced significant results that might be responsible for the obtained results. This might be due to a considerable weight of mean difference derived from lower reported values for mean serum zonulin and small standard deviation. Moreover, the study was conducted on patients with colorectal liver metastasis (CLM) that have higher postoperative levels of serum zonulin rather than colorectal cancer patients without liver metastasis (23). Therefore, there may be a liver barrier playing a role in the change of postoperative zonulin levels in CLM. When we examined the association between study duration and the effect of probiotic/synbiotic on serum levels of zonulin, we found that studies with longer duration of intervention had lower serum levels of zonulin. Some strengths should be highlighted in this meta-analysis. We used a rigorous search strategy and a systematic methodology based on the current guidelines for systematic reviews to determine the effects of supplemental probiotic/synbiotic on serum levels of zonulin, a hallmark of intestinal permeability associated with several chronic conditions. However, several limitations of this study are fully acknowledged. While studies indicated that different species of probiotics act differently in reserving tight junction integrity and barrier function (33), we could not perform subgroup analysis based on probiotic type due to limited number of studies. Furthermore, we could not analyze fecal concentrations of zonulin due to the lack of reported data in this regard. Since the gut is not the only source of circulating zonulin, data on the association of serum levels of zonulin and intestinal zonulin are ambiguous (3). Nevertheless, the decreased ratio of lactulose/mannitol, an indicator of intestinal permeability, was associated with reduced levels of serum zonulin (23).

Conclusion
Oral probiotic supplementation has favorable effects on serum levels of zonulin, a measure of intestinal permeability, although the results should be interpreted with caution due to the influence of a single study on the pooled effect size. High heterogeneity was noted and further evidence is required before definitive recommendations can be made.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.