Acupuncture for Hypertension in Animal Models: A Systematic Review and Meta-Analysis

Objective The aim of this study was to summarize and evaluate the efficacy of acupuncture in hypertension animal study. Methods Studies were searched from six databases, including Medline, Embase, Chinese National Knowledge Infrastructure, Wanfang Data, VIP information database, and Chinese Biomedical Literature Database. Study quality of each included study was evaluated according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, and the risk of bias was evaluated by the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were selected as outcomes. Meta-analyses were performed using Stata 12.0 software. The effect size was calculated by combining SBP/DBP/MAP data with the random effects model, respectively. Results 67 studies containing 1522 animals were included. According to the ARRIVE guideline, 8 items were assessed as poor and 4 items were assessed as excellent. According to the SYRCLE tool, all studies were judged as having high risk of bias. Compared with the hypertension group, the pooled results showed significant antihypertension effects of acupuncture for SBP, DBP, and MAP. Similarly, compared with the sham-acupuncture group, the pooled results showed significant antihypertension effects of acupuncture for SBP, DBP, and MAP. Conclusion Although pooled data suggested that the acupuncture group was superior to the hypertension group or sham-acupuncture group for SBP/DBP/MAP, the presentation of poor methodological quality, high risk of bias, and heterogeneity deserves cautious interpretation of the results.


Introduction
Hypertension is a major modifiable risk factor for cardiovascular and cerebral vascular disease [1,2], affecting about 1.39 billion people worldwide [3]. e main treatment modality for hypertension is pharmacological treatment. Although considerable progress has been made in the field of antihypertensive medicines, only 13.8% of adults with hypertension had their blood pressure (BP) controlled up to the standard worldwide [3]. Because of its relative safety, acupuncture has led to a growing interest among nonpharmacological complementary therapies, in the treatment of hypertension. Also, it has been shown potential in lowering BP, although the effects were not proven sustaining [4].
Plenty of research studies focusing on antihypertensive mechanisms of acupuncture have shown that acupuncture elicits antihypertensive effects through the regulation of renin-angiotensin-aldosterone system, vascular endothelium function, oxidative stress, neuroendocrine system, and so on [5]. However, the efficacy and mechanisms of acupuncture for experimentally induced hypertension have not been systematically investigated yet. e scientific theory for systematic reviews of animal studies was initially summarized in a commentary published in e Lancet [6]. A systematic review of preclinical animal studies contributes to translational medicine and potentially brings about more precise medical care decisions [7]. In addition, systematic review of preclinical evidence could inform the design and contribute to success of future clinical studies, indicate the necessity of further research, reduce unnecessary study replication, and implement the principle of "replacement, refinement, and reduction of animals" in animal research [8]. us, the aim of present review is to synthesize and appraise pooling results of acupuncture's antihypertensive effects in animal models, quantitively assess influencing factors of acupuncture's efficacy, explore future study direction, provide clues for clinical studies, and assess potential publication bias and its influence.

Search Strategy.
e following sources were searched from inception to July 2020: Medline, Embase, Chinese National Knowledge Infrastructure (CNKI), Wanfang Data, VIP information database, and Chinese Biomedical Literature Database. In an effort to identify further published and unpublished research studies, we retrieved the reference lists of relevant reports and review articles identified from electronic databases. Following terms combined were used for searching: acupuncture OR acupoint, hypertension OR blood pressure, animal OR pre-clinical study OR mechanism study.

Inclusion Criteria.
Studies were included if all of the following standards were met: sustaining high blood pressure (HBP) animal model, baseline systolic blood pressure (SBP) ≥ 140 mmHg, diastolic blood pressure (DBP) ≥ 90 mmHg [9,10]; at least one of the following was used as outcome measures: SBP, DBP, and MAP; and SBP/DBP/ MAP was compared with those of hypertension animals receiving sham-acupuncture or no treatment.

Exclusion Criteria.
Studies were excluded if any of the following standards was met: the HBP was induced by stress or adrenaline administration and was not lasting; studies were conducted to compare the effects of different acupuncture methods on hypertension, with no treatment or sham-acupuncture treatment as control; studies did not use BP values as outcomes; and duplicate publications.

Selection of Studies.
Two reviewers screened the titles and/or abstracts of searched studies and excluded obviously irrelevant studies, such as clinical studies, reviews, and nonhypertension and nonacupuncture studies. e full texts were obtained for the remaining studies. A flowchart of study selection is presented in Figure 1.

Data Extraction.
Two reviewers independently assessed the eligible studies and extracted data using a predefined template. e following items were extracted: publication information, experimental animal information, type of animal model, acupuncture manipulation methods, outcome measurements, data of mean outcome, standard deviation, and sample of animals in the acupuncture as well as the control groups. If the results were only shown by chart, the data were obtained by accurate measurement from the figures by software Digxy1.0.0.1. e missing information was sought by sending e-mail or telephone call to the corresponding author of the article. Stata 12.0 software was used for data analysis.

Quality Assessment.
e methodological quality of the included studies was assessed according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, the standards of reporting in animal research. 20 items were included in the guideline. Standards of grading were adjusted based on previous report [11,12]. Items "1" and "11" were marked a lowest score of 0 and a highest score of 1 (0 � inaccurate, not concise, or not reported; 1 � accurate, concise, or reported). e other items (2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, and 20) were marked a lowest score of 0 and a highest score of 2 (0 � clearly inaccurate or not reported; 1 � possibly accurate, unclear, or incomplete; 2 � clearly accurate). e total scores of one study ranged from 0 to 38. e category score was sum of scores by each item. e maximum score was the maximum possible score, i.e., when each item was assessed as clearly accurate [11]. We calculated a ratio quality score/ maximum score, generating three possible quality intervals of which 0.8-1 was considered "excellent," 0.5-0.8 was considered "average," and scores below 0.5 was considered "poor."

Bias Assessment.
e risk of bias was evaluated with the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool [13]. is tool contains 10 items about the selection, performance, detection, attrition, reporting bias, etc. Each item was labeled yes, no, or unclear to score selected articles. Studies with 1 item recognized to be high risk of bias were considered to have an entire high risk of bias. Studies with unclear risk of bias for at least one item were considered to be at unclear risk of bias, and studies with low risk of bias in all items were assessed as low risk of bias [14]. e assessment of the methodological quality and risk of bias was performed by two authors separately. Any divergence was resolved through discussion with a third reviewer.

Statistical
Analysis. Meta-analyses were performed using Stata 12.0 software. Outcome measures of SBP, DBP, and MAP were considered as continuous data. e effect size was calculated by combining these data with the random effects model, respectively. Publication bias was assessed with a funnel plot and Egger's test. e I 2 statistic was used for 2 Evidence-Based Complementary and Alternative Medicine detection of heterogeneity. If the I 2 statistic was higher than 50%, we considered significant heterogeneity was present. To explore the impact of factors potentially influencing the BP outcome, subgroup analyses were conducted for the following factors: acupuncture methods used, age of acupuncture initiation, age of BP measurement, duration of each acupuncture session, total acupuncture sessions, and acupuncture treatment frequency. Sensitivity analyses were performed by deleting one study at a time from the pooled studies.

Results
According to search criteria, we retrieved 4030 potentially relevant records from 6 databases, of which 2012 were duplicate records. After screening titles and abstracts of remaining 2018 records, 1914 records were excluded for one or more of the following reasons: (1) not research studies of hypertension or hypertension combined with other conditions, (2) unsustainable hypertension, e.g., the HBP was caused by cold or electric stress, (3) not animal study, (4) not an acupuncture study or acupuncture combined with other therapy, (5) review articles, and (6) other language. By browsing the whole text of the remaining 104 records, a total of 37 studies were excluded for one or more of the following reasons: (1) unsustainable hypertension, (2) not animal study, (3) without BP measurement, (4) without control group, and (5) full article could not be obtained. Finally, 67 eligible studies were included, and 1522 animals were involved in the meta-analysis.

Subgroup Analysis and Sensitivity Analysis.
To investigate potential factors which influenced the BP measures, we stratified the included studies according to factors, as shown in Tables S1-S5. e subgroup analysis of MAP comparing acupuncture and sham-acupuncture was not conducted due to limited number of studies.
For SBP/DBP/MAP, sensitivity analyses showed that the results did not largely change after omitting any one study (Figures S1-S6).  Table S6.

Discussion
To our knowledge, we present the first systematic review and meta-analysis of the efficacy of acupuncture in  ird, most included studies are defective in terms of randomization, allocation concealment, blinding assessment, and sample size calculation by both assessment scales, which are central for the preclinical study design criteria [81]. Low methodological quality and high risk of bias weakened the robustness of the current preclinical evidence. Fourth, the high level of heterogeneity among different studies   Evidence-Based Complementary and Alternative Medicine is deemed as a prognostic factor independent of BP value for cardiovascular mortality [82,83]. Evidence shows that cardiac accident of hypertension may be the consequence of elevated variability but not BP alone [84,85]. In addition, there is a strong correlation between elevated BPV and aggravated target organ damage [86]. Only 1 study assessed the effect of acupuncture on BPV [58]. Analysis of BPV may provide information about autonomic         function, and there are also studies which demonstrated that acupuncture has the potential to mediate autonomic function [87]. us, exploring the effect of acupuncture on hypertension animal BPV may contribute to finding out the potential efficacy of acupuncture.

Implications.
Preclinical studies usually provide evidence basis for clinical studies. To demonstrate the scientific function pathway of acupuncture, animal studies should be designed, study data should be analysed, and the result should be reported appropriately and transparently. ere is a wealth of evidence showing the poor design of animal research [88], which is an obstacle to advance animal research into promising achievement for human disease. e ARRIVE guidelines have been issued since 2010 to unify the criteria of reporting in animal research and were utilized by this review to assess the quality of paper in respect of acupuncture's antihypertension effects.
e ARRIVE [89] is a reporting guideline containing a 20-entry checklist about each part of publishing papers. e SYRCLE tool was applied to assess the internal validity and risk of bias within individual studies [13]. In addition to items of ARRIVE and SYRCLE, acupuncture's details including acupuncture manipulation, treatment duration, if electroacupuncture is used, the frequency and intensity of pulse method, etc. should be especially noted. Although these assessment tools could be used in acupuncture preclinical study [90], the need for adjusted and unified criteria in reporting of acupuncture preclinical study is imperative. Similar to the Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA) [91], a specialized guideline for acupuncture's preclinical study should be formulated in order to promote study's transparency and repeatability.

Conclusion
To our knowledge, we present the first systematic review and meta-analysis of the efficacy of acupuncture in treating animal models of hypertension. Although pooled data suggested that the acupuncture group was superior to the hypertension group or sham-acupuncture group for SBP/ DBP/MAP, the presentation of poor methodological quality, high risk of bias, and heterogeneity deserves cautious interpretation of the results.
Data Availability e data are available on request to the corresponding author.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
L-Y X contributed to the conception of the study. e manuscript protocol was drafted by L-Y X and ZL. e search strategy was developed by all the authors and was performed by S-Q Y, Y-X Z, and R-Y L. W-L L and H-Y W independently screened the potential studies and extracted data of included studies. Y-Z D and H-Y S assessed methodological quality and the risk of bias. L-Y X completed the data synthesis. X-Y D arbitrated in cases of disagreement, revised the manuscript, and ensured the absence of errors. All authors approved the publication of the work.