==== Front Medicine (Baltimore) Medicine (Baltimore) MEDI Medicine 0025-7974 1536-5964 Lippincott Williams & Wilkins Hagerstown, MD MD-D-20-11269 10.1097/MD.0000000000023730 23730 3400 Research Article Study Protocol Systematic Review Regulatory effect of traditional Chinese medicine on gut microbiota in patients with atherosclerosis A protocol for systematic review and meta-analysisHuang Langlang PhDa Wang Jianan MMb Xu Ri MMb Liu Yanwei PhDb http://orcid.org/0000-0002-4793-7904Liu Zhongyong MMb∗ a Jiangxi University of Traditional Chinese Medicine b The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi Province, PR China. ∗ Correspondence: Zhongyong Liu, Department of Cardiovascular, The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, No.445 Bayi Avenue, Donghu District, Nanchang, Jiangxi 330006, PR China (e-mail: lzyongmail@163.com). 11 12 2020 11 12 2020 99 50 e2373014 11 2020 17 11 2020 Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.2020This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by/4.0Abstract Background: Atherosclerosis is the pathological basis of many cardiovascular and cerebrovascular diseases, and its pathogenesis is complex. Recent studies revealed a significant role of gut microbiota in the onset and development of atherosclerosis. Traditional Chinese medicine has rich clinical experience and unique advantages in the treatment of atherosclerosis. A large number of studies have proved that traditional Chinese medicine has the functions of reducing blood lipid, regulating gut microbiota, and resisting inflammation. The aim of this systematic review is to observe the randomized controlled trial of traditional Chinese medicine in treating gut microbiota, so as to evaluate the effectiveness and safety of traditional Chinese medicine in treating atherosclerosis patients. Methods: The English database (PubMed, Web of Science, Embase, the Cochrane Library) and Chinese database (China National Knowledge Infrastructure, the Chongqing VIP Chinese Science, and Technology Periodic Database, Wanfang Database, and China Biomedical Literature Database) will be searched up to October 2020. We will also manually search the Chinese clinical trial register, conference papers, and unpublished studies or references. Randomized control trials of traditional Chinese medicine treatment of atherosclerosis were collected comprehensively, and 2 researchers will independently screen literature, data extraction, and evaluation the quality of literature methodology. The primary outcomes are lipid metabolism and gut microbiota and their metabolites. The secondary outcomes are the change of inflammatory markers. Meta-analysis was performed by RevMan 5.3.5 software. The Grades of Recommendation, Assessment, Development, and Evaluation will be used to evaluate the outcome quality of evidence. Results: This study will comprehensively review the existing evidence of traditional Chinese medicine in treating atherosclerosis from the perspective of gut microbiota. Conclusion: This study will provide information on the effectiveness and safety of traditional Chinese medicine in treating atherosclerosis from the perspective of gut microbiota. Unique INPLASY number: INPLASY2020110056. Keywords atherosclerosisgut microbiotaprotocolsystematic reviewtraditional Chinese medicineNational Natural Science Foundation of ChinaNo.81660781Zhongyong LiuNational Natural Science Foundation of ChinaNo.81960849Zhongyong LiuScience and Technology Plan Project of Jiangxi Provincial Administration of Traditional Chinese MedicineNo.2019A054Ri XuJiangxi Province 2020 Postgraduate Innovation Special Fund ProjectNo.YC2020-B144Langlang HuangJiangxi Science and Technology Department ProjectNO.20202BAB216034Yanwei LiuOPEN-ACCESSTRUE ==== Body 1 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 cerebrovascular 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] The pathogenesis of AS is unclear, and the risk factors include smoking, drinking, hypertension, hyperlipidemia, and obesity, among which abnormal lipid metabolism is the most critical risk factor.[4–6] This disease's pathogenesis is complex, involving lipid infiltration, endothelial injury, platelet hyperfunction, inflammatory reaction, oxidative stress, and so on.[7] However, 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[9–11]; 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), short-chain 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, multi-target, 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.[14–18] 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] In 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. 2 Methods 2.1 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] 2.2 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. Table 1 PubMed search strategy. Number Search terms #1 Atherosclerosis[MeSH] #2 Atheroscleroses[title/abstract] #3 Atherogenesis[title/abstract] #4 #1 OR #2 OR #3 #5 Gastrointestinal Microbiome[MeSH] #6 Gut Microbiota[title/abstract] #7 Microbiome, Gastrointestinal[title/abstract] #8 Gut Microbiome[title/abstract] #9 Gut Microbiomes[title/abstract] #10 Microbiome, Gut[title/abstract] #11 Gut Microflora [title/abstract] #12 Microflora, Gut[title/abstract] #13 Gut Microbiotas[title/abstract] #14 Microbiota, Gut[title/abstract] #15 Gastrointestinal Flora[title/abstract] #16 Flora, Gastrointestinal[title/abstract] #17 Gut Flora[title/abstract] #18 Flora, Gut[title/abstract] #19 Gastrointestinal Microbiota[title/abstract] #20 Gastrointestinal Microbiotas[title/abstract] #21 Microbiota, Gastrointestinal[title/abstract] #22 Gastrointestinal Microbial Community[title/abstract] #23 Gastrointestinal Microbial Communities[title/abstract] #24 Microbial Community, Gastrointestinal[title/abstract] #25 Gastrointestinal Microflora[title/abstract] #26 Microflora, Gastrointestinal[title/abstract] #27 Gastric Microbiome[title/abstract] #28 Gastric Microbiomes[title/abstract] #29 Microbiome, Gastric[title/abstract] #30 Intestinal Microbiome[title/abstract] #31 Intestinal Microbiomes[title/abstract] #32 Microbiome, Intestinal[title/abstract] #33 Intestinal Microbiota[title/abstract] #34 Intestinal Microbiotas[title/abstract] #35 Microbiota, Intestinal[title/abstract] #36 Intestinal Microflora[title/abstract] #37 Microflora, Intestinal[title/abstract] #38 Intestinal Flora[title/abstract] #39 Flora, Intestinal[title/abstract] #40 Enteric Bacteria[title/abstract] #41 Bacteria, Enteric[title/abstract] #42 #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27 OR #28 OR #29 OR #30 OR #31 OR #32 OR #33 OR #34 OR #35 OR #36 OR #37 OR #38 OR #39 OR #40 OR #41 #43 Medicine, Chinese Traditional[MeSH] #44 Traditional Chinese Medicine[title/abstract] #45 Chung I Hsueh[title/abstract] #46 Hsueh, Chung I[title/abstract] #47 Traditional Medicine, Chinese[title/abstract] #48 Zhong Yi Xue[title/abstract] #49 Chinese Traditional Medicine[title/abstract] #50 Chinese Medicine, Traditional[title/abstract] #51 Traditional Tongue Diagnosis[title/abstract] #52 Tongue Diagnoses, Traditional[title/abstract] #53 Tongue Diagnosis, Traditional[title/abstract] #54 Traditional Tongue Diagnoses[title/abstract] #55 Traditional Tongue Assessment[title/abstract] #56 Tongue Assessment, Traditional[title/abstract] #57 Traditional Tongue Assessments[title/abstract] #58 #43 OR #44 OR #45 OR #46 OR #47 OR #48 OR #49 OR #50 OR #51 OR #52 OR #53 OR #54 OR #55 OR #56 OR #57 #59 randomized controlled trial[Publication Type] #60 randomized[Title/Abstract] #61 placebo[Title/Abstract] #62 #59 OR #60 OR #61 #63 #4 AND #42 AND #58 AND #62 2.3 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. 2.3.2 Type of participants Patients who meet the diagnostic criteria for AS will be included. 2.3.3 Types of interventions The treatment group was treated with TCM or TCM combined with western medicine. 2.3.4 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-α, CRP, IL-6), safety index, and incidence of adverse events. 2.4 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 2.5 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. Figure 1 Flow diagram of literature retrieval. 2.6 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. 2.7 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. 2.8 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. 2.9 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. 2.10 Assessment of heterogeneity The heterogeneity test adopts P ≤ .10 and I2 ≥ 50% as the significance judgment criteria, and when P > .10 and I2 < 50%, the fixed effects models will be used for data analysis. If P ≤ .10 and I2 ≥ 50%, the random effects model will be chosen. 2.11 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, meta-analysis 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. 2.12 Sensitivity analysis We consider sensitivity analysis for methodological quality and test the results’ robustness by excluding the risk of low quality and high bias. 2.13 Subgroup analysis If heterogeneity is observed in the study, we will use subgroup analysis for research. 2.14 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.” 2.15 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. 3 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. Author contributions Conceptualization: Langlang Huang, Zhongyong Liu. Data curation: Langlang Huang, Jianan Wang, Ri Xu. Formal analysis: Jianan Wang, Ri Xu, Yanwei Liu. Investigation: Langlang Huang, Zhongyong Liu. Methodology: Langlang Huang, Jianan Wang, Ri Xu, Yanwei Liu. Software: Jianan Wang, Yanwei Liu. Supervision: Langlang Huang, Zhongyong Liu. Writing – original draft: Langlang Huang, Jianan Wang, Yanwei Liu, Zhongyong Liu. Writing – review & editing: Langlang Huang, Jianan Wang, Ri Xu. Abbreviations: AS = atherosclerosis, BAs = bile acids, GM = gut microbiota, RCT = randomized controlled trial, SCFAs = short-chain fatty acids, TCM = traditional Chinese medicine, TMAO = trimethylamine N-oxide. How to cite this article: Huang L, Wang J, Xu R, Liu Y, Liu Z. Regulatory effect of traditional Chinese medicine on gut microbiota in patients with atherosclerosis: a protocol for systematic review and meta-analysis. Medicine. 2020;99:50(e23730). This study is supported by the National Natural Science Foundation of China (No. 81660781, No. 81960849), Science and Technology Plan Project of Jiangxi Provincial Administration of Traditional Chinese Medicine (No. 2019A054), Jiangxi Province 2020 Postgraduate Innovation Special Fund Project (No. YC2020-B144), Jiangxi Science and Technology Department Project (No. 20202BAB216034). The funders had no role in the design, execution, or writing of the study. The authors have no conflicts of interest to disclose. Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. ==== Refs References [1] Stavnsbo M Resaland GK Anderssen SA Reference values for cardiometabolic risk scores in children and adolescents: suggesting a common standard . Atherosclerosis 2018 ;278 :299 –306 .30477756 [2] Libby P Buring JE Badimon L Atherosclerosis . Nat Rev Dis Primers 2019 ;5 :56 –74 .31420554 [3] The Writing Committee of the Report on Cardiovascular Health Diseases in China . Report on Cardiovascular Health and Diseases in China 2019: an Updated Summary . Chin Circ J 2020 ;35 :833 –54 . [4] Rahman F Al RFM Blaha MJ Relation of diastolic blood pressure and coronary artery calcium to coronary events and outcomes (from the multi-ethnic study of atherosclerosis) . Am J Cardiol 2017 ;120 :1797 –803 .28864316 [5] Schoemaker MH Kleemann R Morrison MC Casein hydrolysate based formulation attenuates obesity and associated non-alcoholic fatty liver disease and atherosclerosis in LDLr-/-. Leiden mice . PLoS One 2017 ;12 :e0180648 .28678821 [6] de Ronde MWJ Kok MGM Moerland PD High miR-124-3p expression identifies smoking individuals susceptible to atherosclerosis . Atherosclerosis 2017 ;263 :377 –84 .28457624 [7] Holdt LM Teupser D Long noncoding RNA ANRIL: Lnc-ing genetic variation at the chromosome 9p21 locus to molecular mechanisms of atherosclerosis . Front Cardiovasc Med 2018 ;5 :145 .30460243 [8] Lässiger-Herfurth A Pontarollo G Grill A The gut microbiota in cardiovascular disease and arterial thrombosis . Microorganisms 2019 ;7 :691 . [9] Ryan KK Tremaroli V Clemmensen C FXR is a molecular target for the effects of vertical sleeve gastrectomy . Nature 2014 ;509 :183 –8 .24670636 [10] Elce A Amato F Zarrilli F Butyrate modulating effects on proinflammatory pathways in human intestinal epithelial cells . Benef Microbes 2017 ;8 :841 –7 .28856908 [11] Chistiakov DA Bobryshev YV Kozarov E Role of gut microbiota in the modulation of atherosclerosis-associated immune response . Front Microbiol 2015 ;6 :671 .26175728 [12] Din AU Hassan A Zhu Y Amelioration of TMAO through probiotics and its potential role in atherosclerosis . Appl Microbiol Biotechnol 2019 ;103 :9217 –28 .31655880 [13] Ascher S Reinhardt C The gut microbiota: an emerging risk factor for cardiovascular and cerebrovascular disease . Eur J Immunol 2018 ;48 :564 –75 .29230812 [14] Wang F Zhao CY Tian GF Naringin alleviates atherosclerosis in ApoE-/- Mice by regulating cholesterol metabolism involved in gut microbiota remodeling . J Agric Food Chem 2020 ;68 :12651 –60 .33107729 [15] Zhu BR Zhai Y Ji MJ Alisma orientalis beverage treats atherosclerosis by regulating gut microbiota in ApoE mice . Front Pharmacol 2020 ;11 :570555 .33101028 [16] Song N Jia LQ Cao HM Gypenoside inhibits endothelial cell apoptosis in atherosclerosis by modulating mitochondria through PI3K/Akt/Bad pathway . Biomed Res Int 2020 ;2020 :2819658 .32685460 [17] Liu B Song ZY Yu JP The atherosclerosis-ameliorating effects and molecular mechanisms of BuYangHuanWu decoction . Biomed Pharmacother 2020 ;123 :109664 .31887542 [18] Wang H Zhong L Mi SH Tanshinone IIA prevents platelet activation and down-regulates CD36 and MKK4/JNK2 signaling pathway . BMC Cardiovasc Disord 2020 ;20 :81 .32059638 [19] Chen ML Long Y Yong Z Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota . mBio 2016 ;7 :e02210 .27048804 [20] Yang G He HQ Chen G Effect of traditional Chinese medicine in attenuating coronary heart disease and main risk factors by regulating gut micro-biota . Chin J Chin Mater Med 2020 ;45 :29 –36 . [21] Fang JM Wang QL Li L Research progress of traditional Chinese medicine in the prevention and treatment of vascular diseases by regulating gut microbiota . World Sci Technol-Mode Tradit Chin Med 2019 ;21 :1127 –34 . [22] Shamseer L Moher D Clarke M Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation . BMJ 2015 ;350 :1 –25 . [23] Higgins JP Altman DG Gotzsche PC The Cochrane Collaboration's tool for assessing risk of bias in randomised trials . BMJ 2011 ;343 :1 –9 . [24] Deng T Wang Y Huang D Methods for formulating clinical practice guidelines: GRADE method theory . Chin J Evid Based Cardiovasc Med 2018 ;10 :1441 –5 .