The Effects of Probiotic Supplementation on Glycemic index, Lipid Profiles and Inflammatory cytokines in NAFLD Patients: A Protocol for a Systematic Review and Meta-Analysis

Introduction

Non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver disease worldwide. In an aggressive type, non-alcoholic steatohepatitis (NASH) might lead to cirrhosis and hepatocellular carcinoma progression. Currently, there is no cer-tified drug applied to treat NASH [1]. The pathogenesis path of NASH is yet remained to be known. However, the underlying factors mentioned below could be taken into account: obesity, high-calorie/high-fat diet, diabetes, hypercholesterolemia, and cer- tain drugs either [2]. Over this issue, efforts done to alter people’s lifestyles have been suggested. Still, they were hardly sustainable, so it necessitates to conduct investigations through some new ther¬apeutic strategies, including weight reduction and diet changes, which are recommended as the first step involved in patients’ treat¬ment procedure under such conditions [3].

Once weight loss is obtained and appropriately sustained, it may gradually improve NAFLD(1). Although there is no recognized medication or surgical method approved for the treatment of NA¬FLD, countless numbers of clinical trials have demonstrated sev¬eral pharmacological treatments suitable to cure NAFLD/NASH, such as diabetes medications, lipid-lowering drugs, antioxidants, and anti-tumor necrosis factor (TNF)-α agents [4].

The mechanisms related to the development and progression of NAFLD are yet to be fully understood. This process is assumed to be the substantial consequence of the interactions amongst progressive environment-driven epigenetic code changes, genet¬ic background, and molecular alterations. Environmental factors such as unhealthy diet and the appearance of a stagnant seden-tary routine, which drive epigenomic reprogramming of the host genome via post-translational modifications of gene expression, could ultimately evolve phenotypic changes in the organism [5]. Seamlessly, obesity is thought to be the most vital risk factor af-filiated with various NAFLD series in which abdominal fat is cor-related with a volume of steatosis observed on liver biopsy(1) with a striking increased risk of insulin resistance and emerging multi-ple diseases in children as in adults [6].

NAFLD often gives rise to dyslipidemia in which increased se¬rum TG and LDL-C levels and decreased HDL-C levels could be demonstrated [7]. Additionally, recent evidence suggested an in-teraction between the liver and gut called the ‘gut-liver axis’ might participate in the evolution of phenotypic replacement from NAFL to a much more severe state like NASH NASH-related fibrosis. In-deed, NAFLD is associated with escalating intestinal permeability (IP) and small intestinal bacterial overgrowth (SIBO) in humans, all connected to the severity of hepatic steatosis. Hence, several studies have introduced gut microbiota modulation via probiotics, prebiotics, and synbiotic as an efficacious solution to improve the obesity state and subsequent NAFLD(5).

The World Health Organization considers probiotics as live micro-organisms that positively impact the intended host as long as ad-ministered in sufficient volumes. The prophylactic and therapeutic impacts of these microorganisms, including the equilibration of intestinal microbiota, diminution in cholesterol levels, hyperten-sion enhancement, diabetes, lactose intolerance, gastrointestinal diseases, immune system improvement, and also plummeting the risk of various cancers, have been reported in different trials [7]. Administration of Lactobacillus and Bifidobacterium probiotic strains, prebiotics, synbiotics (blend of probiotics and prebiotics), and their fusion with nutraceuticals have been represented to be advantageous by dwindling the hepatic triglyceride content, the hepatic tissue inflammation, total body and visceral adipose tissue weight, and also by improving the insulin sensitivity in various animal models suffering NAFLD and have a deeply profound ef¬fect on NASH either [4]. In addition to this, probiotics can make a decline in the activity of Jun N-terminal kinase and nuclear factor κb and also make an increase in the number of natural killer T cells located in the liver. However, other conducted animal studies could not demonstrate an impact on hepatic steatosis and necro-inflammation improvement. Probiotics prevented liver fibrosis through the exertion of several alterations over the expression of TGFê?µ. It remains unclear whether probiotics are beneficial in hu¬man patients suffering NASH [3].

Due to the lack of appropriate studies and the emerging require¬ments for further illustration over the effects of probiotics on the treatment of NAFLD and NASH-related disorders in humans, in this study, we seek to evaluate and provide evidence related to the effects of probiotics on the treatment of NAFLD.

Methods

Data sources and selection strategy

We will search the following databases: Medline, EMBASE, Co¬chrane Library, and Web of Science from inception to February 2021. Search terms are keywords and medical subject headings related to NAFLD, NASH, fatty liver, probiotics, prebiotic, symbi¬otic, glycemic index, inflammation, and dyslipidemia. 2 research¬ers will determine the search strategy after several pre-searches.

Inclusion and exclusion criteria

Only randomized controlled trials (RCTs) will be included in this study and related to participants, all adult patients who met the ac¬cepted diagnostic criteria for NAFLD will be included in this study without discrimination about sex or ethnic origin In the case of interventions, only RCTs comparing probiotics and supplementa¬tion with placebo will be included in this study. Also, the minimum duration of treatment is eight weeks. The exclusion criteria will include other causes of hepatic steatosis like hepatitis B, hepatitis C, and genetic liver infection, such as Wilson’s illness and hemo-chromatosis.

Outcomes

The trials should have measured at least one of the following items:

Glycemic outcomes

(1) Differences in glycated hemoglobin

(2) Differences in fasting blood glucose

(3) Differences in fasting insulin

(4) Differences in homeostasis model assessment of insulin resis¬tance

Lipidomic outcomes

(1) Differences in High-density lipoprotein

(2) Differences in Low-density lipoprotein

(3) Differences in Total triglyceride

(4) Differences in Total cholesterol

Inflammatory outcomes

(1) Differences in IL6

(2) Differences in IL1β

(3) Differences in TNFα

(4) Differences in CRP

Data selection

Two reviewers will freely screen the literature included in the me-ta-analysis according to the eligibility criteria listed in this study. The title and abstract will be read first in the literature selection process. The full text will be further read to determine the final inclusion after excluding the apparent irrelevant literature. Two other reviewers will independently assess the included studies’ re¬porting quality. The process of literature identification and screen¬ing is shown in Figure 1.

             Figure 1: Studies selection strategy

Data extraction and methodological quality

Using a predefined data extraction sheet from all studies that meet the eligibility criteria, data will be extracted by two reviewers in¬dependently as follows:

- Basic information, including study design, the author first and last name, country, publication year, sample size, and grouping;

- Characteristics of the participant including age, sex, race, and the number of participants.

-Methodological approach, like randomized method, blinding method, and assessed risk of bias; - Time and type of interventions and controls;

- Outcomes, including glycemic, lipidomic, and inflammatory out-come indicators, as well as adverse events.

A third reviewer will check the process of data extraction for con¬firmation. Besides, on the off chance that there’s any contradiction in data extraction, it has to be re-examined by the third reviewer. We will contact the corresponding authors and send a request for additional missing necessary data. The study will be deleted if the lost data is unavailable.

Risk of bias

Two reviewers will assess the study’s quality, which will be based on the following items:

- Random allocation method;

- The allocation scheme is hidden;

- Blind methods are used for subjects and treatment planners;

- Blind method for measurement results;

- The integrity of the resulting data;

- Selectively report the results of the study;

- Other sources of bias.

Studies with a low risk of bias were considered high quality. If there were any conflicts while assessing the quality of data, they would be settled through conversation and discussion.

Statistical analysis

We will analyze results qualitatively and quantitatively. The me-ta-analysis will be performed using the Stata software, and P-value < 0.05 will be statistically significant. The relative risk and the 95% confidence intervals will be used for the outcomes. We will calculate effect size as Standardized Mean Difference (SMD) and 95% CI to evaluate the effects of probiotics on variables. Statisti¬cal heterogeneity will be measured using the Q and the Higgins I2 statistics. We will perform subgroup analysis according to the type of sample, different dosages of probiotics, and duration treat¬ment to identify potential heterogeneity sources amongst included studies in the meta-analysis. A fixed-effect model or random-ef-fects model will be employed to assess the difference based on statistical homogeneity between the studies (I2_50% or P>.10) or statistical heterogeneity between the studies (I2≥50% or P<.10). The possibility of publication bias will be assessed with the Begg and the Egger regression test as statistically and also funnel plot as graphically

Discussion

Over the past decade, the number of studies evaluating the effect of probiotics on NAFLD patients has increased and provided data related to improving glycemic, lipidomic, and inflammatory con¬ditions (8). This systemic review and meta-analysis aim to pre¬cisely estimate the impacts of probiotics supplementation on gly¬cemic, lipidomic, and inflammatory factors in NAFLD patients. To determine and interpret probiotic effect on improving NAFLD condition, we will evaluate and perform further subgroup analy¬sis related to probiotics’ efficacy and safety and different duration treatment time. There will be several strengths in our systemat¬ic review and meta-analysis. Our search strategy is very detailed and spanned multiple conditions in NAFLD. We will also perform subgroup analysis to explore the possible sources of the heteroge¬neity, and attempts will be made to select studies from different countries, which will provide more generalizable results. In addi¬tion data screening and extraction, the quality assessment will be performed independently by two reviewers. We hope that this sys¬tematic review and meta-analysis provide evidence assessing the effectiveness and safety of probiotics supplementation in NAFLD regarding glycemic, lipidomic, and inflammatory factors.

Ethical Approval and Consent to participate

Not applicable

Consent for publication

Not applicable

Availability of supporting data

Data sharing not applicable to this article as no datasets were gen¬erated or analyzed

Competing interests

The authors declare that there is no conflict of interest

Funding

No funding to declare

Authors’ contributions

SM, PRM and GP: provided the conception and design of the study. SM and HH provide drafting the article and preliminary search. SE and MZ revised it critically, and final approval of the version to be submitted; GP: writing – review & editing.

Acknowledgments

We thank the Department of clinical biochemistry of Tehran uni¬versity of medical sciences colleagues for their support during this research.

References

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