A protocol for systematic review and meta-analysis

Background:Type 2 diabetes (T2DM), which is themajor type of diabetes, accounts for more than 90%of all case of diabetes, and its pathogenesis remains inconclusive. Recent studies have revealed a significant role of gut microbiota in the onset and development of T2DM. Traditional Chinese medicine (TCM) has accumulated rich clinical experience in the treatment of T2DM for thousands of years and a large amount of studies have shown that TCM has the capacity of lowering blood glucose andmodulating gut microbiota. The aim of this systematic review is to evaluate all randomized controlled trials on TCM for gut microbiota to assess the effectiveness and safety of TCM in T2DM patients. Methods: Seven electronic databases (Web of Science, PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang Database, and VIP Information-Chinese Scientific Journal Database) will be searched from inception to present in the English and Chinese languages. Eligible randomized controlled trials evaluating the effect of TCM in T2DM patients, compared with western medicine, placebo or no intervention will be included in the study. The primary outcomes are the glucose metabolism and gut microbiota as well as its metabolites. The second outcomes are changes in weight, and changes in inflammatory markers. Two authors will independently select studies, extract data, and assess the quality of the studies by scanning the titles, abstracts, and full texts. The meta-analysis will be conducted using ReviewManager version 5.3. The results will be presented as risk ratios for dichotomous data and adverse events, and as mean differences for continuous data. Result: The study will provide a summary of current evidence for the treatment of T2DM from the perspective of gut microbiota by using TCM based on the outcome measures. Conclusion: The systematic review will evaluate the efficacy of TCM in treating T2DM from the perspective of gut microbiota, providing current evidence and laying a foundation for further work in the field. PROSPERO registration number: CRD42020188043. Abbreviations: CHM =Chinese herbal medicine, RCT = randomized controlled trial, ROB= risk of bias, SCFA= short-chain fatty acid, TCM = traditional Chinese medicine, T2DM = type 2 diabetes.


Introduction
Atherosclerosis (AS) is a chronic inflammatory disease related to lipid accumulation and changes in a blood vessel wall components, which occurs in large and medium arteries.It is the pathological basis of many cardiovascular and cerebrovas-cular diseases such as ischemic heart disease, cerebral infarction, and cerebral hemorrhage.With the development of the social economy and the aggravation of population aging, its incidence rate increases, which causes higher mortality and social pressure in the world. [1]According to statistics, about 17 to 18 million people die of atherosclerotic cardiovascular disease every year in the world, accounting for 31% of all deaths; In China, the number of cardiovascular disease patients reached 300 million. [2,3][6] This disease's pathogenesis is complex, involving lipid infiltration, endothelial injury, platelet hyperfunction, inflammatory reaction, oxidative stress, and so on. [7]owever, neither theory can fully explain the formation mechanism of AS alone.
There are more than 3,500 kinds of gut microbiota (GM) in normal human intestines, with a total of about 100 trillion.GM's stable composition and function play an essential role in maintaining the intestinal tract's normal physiological part and immune defense. [8]With the deepening of related research, more and more evidence shows that GM imbalance can lead to the formation of AS by participating in the regulation of cholesterol metabolism, oxidative stress, and inflammation [9][10][11] ; Moreover, GM can also participate in the metabolism of nutrients such as protein, dietary fiber, and choline in vivo, and produces metabolites such as trimethylamine N-oxide (TMAO), shortchain fatty acids), and bile acids (BAs) to regulate the immunity and metabolism of host, thus affecting the occurrence and development of AS. [12,13] Traditional Chinese medicine (TCM), as the crystallization of Chinese conventional wisdom, has multi-component, multitarget, and multi-channel characteristics and plays a unique role in clinical prevention and treatment of diseases.Many modern pharmacological studies have proved that TCM has the functions of protecting vascular endothelial cells, resisting platelet aggregation, reducing blood lipid, resisting inflammation, resisting oxidative stress, and regulating GM. [14][15][16][17][18] After oral administration of TCM, its practical components are mainly absorbed in the intestinal tract, and stay in the intestinal tract for a long time, which is beneficial to regulating GM diversity and metabolism.Moreover, TCM can also play an anti-AS role by regulating the metabolites of GM, such as reducing the production of TMAO and increasing the ratio of lactic acid bacteria bifidobacteria to promote BA metabolism and slow down the AS process. [19]n recent years, although many reviews have summarized the effects of TCM on GM and AS and related clinical trials and basic research have been carried out, [20,21] there is no systematic review of the effects of TCM on AS from the perspective of GM regulation.The purpose of this study is to systematically review randomized controlled trials (RCTs), so as to evaluate the evidence of regulating effect and curative effect of TCM on GM of patients with AS.

Protocol and registration
The protocol has been registered on the INPLASY website, and the registration number is INPLASY2020110056 (URL https:// inplasy.com/inplasy-2020-11-0056/).This report will be performed by the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols. [22]

Literature search
We will search the following databases from their inception onwards to the October 2020: PubMed, Web of Science, Embase, the Cochrane Library, China National Knowledge Infrastructure, the Chongqing VIP Chinese Science and Technology Periodical Database, Wanfang Database, and China Biomedical Literature Database.We will also manually search the Chinese Clinical Trial Register, conference papers, and unpublished studies or references.The search strategy for PubMed is listed in Table 1, a similar method will be applied to the Chinese database.

Inclusion criteria
According to the Participants' principle, Intervention, Comparison, and Outcome, the following standards are formulated.
2.3.1.Type of studies.RCTs on TCM intervention in treating AS patients will be included in this review.

Type of participants.
Patients who meet the diagnostic criteria for AS will be included.

Types of interventions.
The treatment group was treated with TCM or TCM combined with western medicine.

Type of comparators.
The control group will receive western medicine treatment or without intervention and was not treated with TCM.
2.3.5.Types of outcome measures.The lipid metabolism outcome indicators (TG, TC, HDL-C, LDL-C, HDL, LDL, ApoA1, ApoB) and GM and its metabolites (GM structure and diversity, TMAO, short-chain fatty acids, BA) are the primary outcome indicators of this study.This study's secondary outcome include carotid intima-media thickness, atherosclerotic plaque area, inflammatory factor level (such as TNF-a, CRP, IL-6), safety index, and incidence of adverse events.

Exclusion criteria
(1) TCM combined with other non-drug adjuvant therapies (such as tai chi, acupuncture, and moxibustion); (2) Documents with similar original data and repeated publication; (3) Documents for which accurate data cannot be obtained; (4) Case report, conference papers, and summaries; (5) No RCT

Studies selection
Two researchers in the research group independently searched out the literature.First, identical pieces of literature were screened and excluded by Endnote X9 document management software.The summary of the title exclusion system was read, and finally, the full text was read, which met the inclusion criteria.In case of disagreement, the third author discussed and decided together.The selection process will be shown according to the Preferred Reporting Items for Systematic Review and Meta-Analysis flow chart in Figure 1.

Data extraction and management
After the literature was included, 2 other researchers independently completed the data extraction, including the author's name, year of publication, title, country, average age, gender, study design, the total number of cases, participants, intervention measures, comparison, outcome, and any other relevant information.

Quality assessment of the studies
According to the bias risk evaluation standard of randomized controlled trials provided by Cochrane Handbook, [23] the literature quality evaluation is carried out, which includes the following 6 aspects: random sequence generation, allocation concealment, blinding of participants, caregivers, outcome assessors, incomplete outcome data, selective outcome reporting, and other bias.According to the specific scoring rules, the 2 researchers evaluated 3 types: "low risk," "high risk," and "uncertain risk."If there is any difference in quality evaluation, discuss it with the third author.

Measures of treatment effect
According to the different variables, the dichotomous variables are expressed as risk ratio and 95% confidence intervals.Continuous variables are defined as weighted mean difference or standardized mean difference and 95% confidence intervals.

Management with missing data
If there are insufficient or lost data, we will first contact the original author by email or telephone.If the lost data cannot be obtained, we will discard the unusable data and only analyze the available data.

Assessment of heterogeneity
The heterogeneity test adopts P .10 and I 2 ≥ 50% as the significance judgment criteria, and when P > .10 and I 2 < 50%, the fixed effects models will be used for data analysis.If P .10 and I 2 ≥ 50%, the random effects model will be chosen.

Data synthesis
RevMan5.3.5 software will be used for this meta-analysis.
According to the heterogeneity level included in the study, the fixed-effect model or the random effect model was selected.We will conduct a meta-analysis of at least 3 qualified standards.Otherwise, if only 1 or 2 studies met the inclusion criteria, metaanalysis will not be undertaken, but the descriptive analysis will be adopted.If more than 10 articles are included, the inverted funnel diagram is used to analyze publication bias.

Sensitivity analysis
We consider sensitivity analysis for methodological quality and test the results' robustness by excluding the risk of low quality and high bias.
Table 1 PubMed search strategy.

Number
Search terms

Subgroup analysis
If heterogeneity is observed in the study, we will use subgroup analysis for research.

Summary of evidence
The evidence quality of each result is evaluated by the method of Recommendation, Assessment, Development, and Evaluation. [24]The evaluation will be divided into 4 qualities: "very low," "low," "medium," or "high."

Ethical approval and dissemination
Ethical approval is not necessary for this study because there is no individual data will be used.The research results will be published in a peer-reviewed journal and conference presentations.

Discussion
AS belongs to the category of "Mai Bi" in TCM.The theory of TCM holds that the interior and exterior of the heart and intestine, so it is possible to treat heart diseases by regulating GM.
TCM has less toxic and side effects in the prevention and treatment of AS.With the development of sequencing technology and metagenome research, many studies have confirmed that TCM can regulate GM and its metabolites and resist AS.However, there is no systematic evaluation of RCTs on the role of TCM in AS treatment from the perspective of GM.Therefore, we hope that this study can provide the latest evidence for the effectiveness and safety of TCM in treating AS and regulating of GM and its metabolites and being used to guide clinical practice.