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

Authors: Ling-Yong Xiao; Zheng Li; Yu-Zheng Du; Hui-Yan Shi; Si-Qi Yang; Yue-Xin Zhang; Rui-Yu Li; Wan-Ling Lin; He-Yang Wang; Xiao-Yu Dai
Journal: Evidence-Based Complementary and Alternative Medicine
 (2021)

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## Abstract

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.


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## Body

## 1. Introduction

Hypertension is a major modifiable risk factor for cardiovascular and cerebral vascular disease [1, 2], affecting about 1.39 billion people worldwide [3]. The 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. The scientific theory for systematic reviews of animal studies was initially summarized in a commentary published in The 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]. Thus, 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.


## 2. Materials and Methods

### 2.1. Search Strategy

The 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.


### 2.2. 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.


### 2.3. 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.


### 2.4. 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. The full texts were obtained for the remaining studies. A flowchart of study selection is presented in Figure1.Figure 1
Flowchart of the study selection.


### 2.5. Data Extraction

Two reviewers independently assessed the eligible studies and extracted data using a predefined template. The 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. The 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.


### 2.6. Quality Assessment

The 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). The 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). The total scores of one study ranged from 0 to 38. The category score was sum of scores by each item. The 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.”


### 2.7. Bias Assessment

The risk of bias was evaluated with the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool [13]. This 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].The 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.


### 2.8. Statistical Analysis

Meta-analyses were performed using Stata 12.0 software. Outcome measures of SBP, DBP, and MAP were considered as continuous data. The 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. TheI2 statistic was used for detection of heterogeneity. If the I2 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.


## 2.1. Search Strategy

The 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.


## 2.2. 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.


## 2.3. 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.


## 2.4. 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. The full texts were obtained for the remaining studies. A flowchart of study selection is presented in Figure1.Figure 1
Flowchart of the study selection.


## 2.5. Data Extraction

Two reviewers independently assessed the eligible studies and extracted data using a predefined template. The 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. The 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.


## 2.6. Quality Assessment

The 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). The 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). The total scores of one study ranged from 0 to 38. The category score was sum of scores by each item. The 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.”


## 2.7. Bias Assessment

The risk of bias was evaluated with the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool [13]. This 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].The 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.


## 2.8. Statistical Analysis

Meta-analyses were performed using Stata 12.0 software. Outcome measures of SBP, DBP, and MAP were considered as continuous data. The 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. TheI2 statistic was used for detection of heterogeneity. If the I2 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.


## 3. 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.


### 3.1. Study Characteristics

Totally, 1522 animals were included for comparison. Of the hypertension models in these studies, 63 used SHR, 1 used AngII infusion [44], and 3 used renal artery stenosis [17, 21, 22]. 3 studies simultaneously assessed SBP/DBP/MAP as the outcome measures [42, 48, 52]. 20 studies assessed SBP and DBP as the outcome measures [23, 32, 35, 36, 38, 41, 43, 45, 47, 49, 50, 53, 55, 56, 60, 62, 68, 72, 79, 80]. 3 studies assessed SBP and MAP as the outcome measures [37, 58, 70]. 35 studies assessed only SBP as the outcome measures [9, 17–22, 24–27, 29–31, 33, 34, 39, 40, 46, 51, 54, 57, 59, 61, 63–65, 67, 69, 71, 73, 75–78]. 6 studies assessed only MAP as the outcome measure [15, 16, 28, 44, 66, 74]. 40 studies adopted manual acupuncture [9, 17, 20, 25, 29, 32–41, 43, 45–47, 50, 51, 53, 54, 57–63, 65–68, 70, 71, 75–78], of which 35 adopted manipulation instead of simple needle penetration [9, 17, 20, 25, 29, 32–41, 43, 45–47, 50, 51, 53, 54, 57, 59–61, 63, 65, 66, 68, 71, 76–78]. 20 studies used EA as intervention [15, 16, 18, 21–23, 27, 28, 30, 31, 42, 44, 48, 52, 64, 69, 72–74, 79]. 4 studies combined manipulation and EA as intervention [24, 49, 55, 56]. 2 studies used other auxiliary devices combined with acupuncture as treatment [19, 80]. The needle retaining time ranged from 0.5 minute to 30 minutes. The duration of acupuncture ranged from 1 time to 48 times. Animal age of treatment initiation ranged from 8 weeks to 34 weeks. Study characteristics are shown in Table 1.Table 1
Characteristics of included studies.
StudyAnimals (species, sex, weight)ModelControl groupOutcome measurementWeek-age of acupuncture initiation (weeks)Week-age of BP measurement (weeks)Acupuncture methodsAcupointsAcupuncture retaining time (minutes)Acupuncture duration (sessions)Chen 2001 [15]SHR, male and female, 189.17 ± 31.26 gSHRHTNMAP1013EAST361512Chen 2002 [16]SHR, male and female, NRSHRHTNMAP1114EAST361515Liu 2003 [17]Wistar, NR, 200 ± 20 gRenal ischemiaHTNSBP2224ManipST36, SP3, PC6, KI12014Su 2003 [18]SHR, male, NRSHRHTNSBP922EAST36, LI113045Yang 2006 [19]SHR, male, 270 ± 10 gSHRHTNSBP1215Other deviceST36, LR3315Yang 2008 [20]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBPNRNRManipLR357Wei 2008 [21]SD, male and female, 190 ± 10 gRenal ischemiaHTNSBPNRNREALI112028Qian 2008 [22]SD, male and female, 175 ± 25 gRenal ischemiaHTNSBPNRNREALI112056Liu 2009 [23]SHR, male, 220 ± 20 gSHRHTN, Sham-acuSBP, DBPNRNREAHT7301Jiang 2010 [24]SHR, male, 150 ± 50 gSHRHTNSBP3437EALI11, GB20, SP61518Wang 2010 [25]SHR, male, 200 ± 20 gSHRHTNSBP910ManipLR357Xiong 2011 [26]SHR, male, NRSHRHTNSBP1321EAST36NR40Wang 2011 [27]SHR, male, 200 ± 20 gSHRHTNSBPNRNREALR357Tian 2012 [28]SHR, male, 220 ± 30SHRHTNMAP816EAST36, DU202028Duan 2013 [29]SHR, male, 200 ± 20 gSHRHTNSBP1113ManipLR32014Chen 2013 [30]SHR, male, 385.22 ± 21.26SHRHTNSBP2430EAST36, LI112042Wang 2013 [31]SHR, male, 360 ± 20 gSHRHTNSBP2632EAST36, LI112042Xu 2014 [32]SHR, male, 260 ± 20SHRHTNSBP, DBP1216ManipLR31024Yu 2014 [33]SHR, male, 210 ± 10SHRHTN, Sham-acuSBP1213ManipLR3, KI357Shen 2015 [34]SHR, male, 245 ± 25SHRHTNSBP1721ManipST9120Liu 2015 [35]SHR, male, 215 ± 25SHRHTNSBP, DBP1721ManipST9120Liu 2015 [36]SHR, male, 215 ± 25SHRHTNSBP, DBP1721ManipST9120Zeng 2015 [37]SHR, male, 255 ± 5SHRHTNSBP, MAP1315ManipLR30.512Tian 2015 [38]SHR, male, 225 ± 25SHRHTNSBP, DBP1216ManipLR31024Sun 2015 [39]SHR, male, 225 ± 25SHRHTNSBP1216ManipLR31024Zhang 2015 [40]SHR, male, 210 ± 10SHRHTN, Sham-acuSBP1213ManipKI357Fu 2015 [41]SHR, male, 300 ± 10 gSHRSham-acuSBP, DBP1822ManipST9128Chang Lee 2016 [42]SHR, male, NRSHRHTN, Sham-acuSBPNRNREALR33021Chen 2016 [43]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBP, DBP1011ManipLR357Tan 2016 [44]SD, male, 190 ± 10 gAngII-inducedHTNMAPNRNREALI112014Wang 2016 [45]SHR, male, 250 ± 20 gSHRHTN, Sham-acuSBP, DBP1114ManipST9221Wang 2016 [46]SHR, male, 210 ± 10SHRHTN, Sham-acuSBP1213ManipKI357Wang 2016 [47]SHR, male, 250 ± 20SHRHTNSBP, DBP1115ManipST36, LR32028Xin 2016 [48]SHR, male, 255 ± 15SHRHTNSBP, DBP, MAP1220EAPC63056Wang 2017 [49]SHR, male, 260 ± 9.04SHRHTNSBP, DBP, MAP1216Manip + EAST36, LI111620Zhang 2017 [50]SHR, male, 190 ± 10 gSHRHTNSBP, DBPNRNRManipLR3, PC63015Guo 2017 [51]SHR, male, 190 ± 10 gSHRHTNSBP913ManipST92028Xin 2017 [52]SHR, male, 255 ± 15 gSHRHTNSBP, DBP, MAP1321EAPC63056Xin 2017 [53]SHR, male and female, 220 ± 20 gSHRHTNSBP, DBPNRNRManipST36, LI113021Guo 2018 [54]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBP1010ManipST9301Wang 2018 [55]SHR, male, 259.17 ± 9.71SHRHTNSBP, DBP1216Manip + EAST36, LI111520Wang 2018 [56]SHR, male, NRSHRHTN, Sham-acuSBP, MAP1214MAST36, LR31514Wang 2018 [57]SHR, male, 225 ± 25 gSHRHTNSBP913ManipLR32024Wang 2018 [58]SHR, male, 259.17 ± 9.71SHRHTNSBP, DBP1216Manip + EAST36, LI111520Deng 2018 [59]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBP1216ManipKI3524Huang 2018 [60]SHR, male, 235 ± 15 gSHRHTN, Sham-acuSBP, DBP1115ManipLR3, KI31524Ma 2018 [61]SHR, male, 190 ± 10 gSHRHTNSBP913ManipST92024Dong 2018 [62]SHR, male, 295 ± 15SHRHTNSBP, DBP1725MAST36, ST9, LI112048Zhao 2018 [63]SHR, male, 190 ± 10 gSHRHTNSBP1014ManipST92028Zheng 2018 [64]SHR, male, 275 ± 25 gSHRHTNSBP1721ManipST92028Li 2018 [65]SHR, male, 240 ± 10SHRHTN, Sham-acuSBP1216ManipST36, PC6, BL15, BL203028Luo 2018 [66]SHR, male, 190 ± 10 gSHRHTN, Sham-acuMAP1216EAST36, DU202028Cui 2019 [67]SHR, male, 240 ± 20 gSHRHTNSBP10-1118-19MAST36, ST7, LI113040Luo 2019 [9]SHR, male, 230 ± 20 gSHRHTN, Sham-acuSBP1216ManipLR3524Wang 2019 [68]SHR, male, 235 ± 15 gSHRHTNSBP1216EADU20, LR32030Wang 2019 [69]SHR, male, 280 ± 8 gSHRHTN, Sham-acuSBP, DBP1314ManipLR3107Ma 2019 [70]SHR, male, NRSHRHTNSBP, MAP1214MALR32014Ji 2019 [71]SHR, male, 215 ± 15 gSHRHTNSBP1115ManipDU20, LR32028Zheng 2019 [72]SHR, male, 275 ± 25 gSHRHTNSBP, DBP1721EAST72028Ning 2019 [73]SHR, male, 300 ± 30 gSHRHTNSBP1628EABL231542Sun 2019 [74]SHR, male, 275 ± 25 gSHRHTNMAP1721EAST72028Cui 2020 [75]SHR, male, 180 ± 20 gSHRHTNSBP1014MAST36, ST7, LI113020Hao 2020 [76]SHR, male, 220 ± 30 gSHRHTNSBP1012ManipLR32014Fang 2020 [77]SHR, male, 200 ± 20 gSHRHTNSBP913ManipST36, LI4, LR3528Sun 2020 [78]SHR, male, 220 ± 30 gSHRHTNSBP1012ManipLR32015Sun 2020 [79]SHR, male, 275 ± 25 gSHRHTNSBP, DBP1721EAST72028Yang 2020 [80]SHR, male, NRSHRHTNSBP, DBP1620OtherLR3120NR: not reported; SHR: spontaneously hypertension rat; HTN: hypertension; Sham-acu: sham-acupuncture; SBP: systolic blood pressure; DBP: diastolic blood pressure; MAP: mean arterial pressure; EA: electroacupuncture; MA: manual acupuncture; Manip: manipulation.


### 3.2. Quality Assessment

Twenty items were evaluated according to the ARRIVE guidelines. According to ratio quality score/maximum score, the items 5/6/10/12/13/14/17/18 were considered to be poor. The items 2/3/4/7/9/11/19/20 were considered to be average. Also, the items 1/8/15/16 were considered to be excellent. Detailed information of identified studies’ quality is shown in Table2.Table 2
Quality assessment according to the Animal Research: Reporting In Vivo Experiment (ARRIVE) guidelines.
StudiesARRIVE items1234567891011121314151617181920TotalChen 2001 [15]0111010200010022011215Chen 2002 [16]1111010100010022001214Liu 2003 [17]0111010101010102011012Su 2003 [18]1111010100000122001214Yang 2006 [19]1111011201011122012019Yang 2008 [20]1211011101012222011222Wei 2008 [21]1201011111010122111018Qian 2008 [22]1101010101010122001215Liu 2009 [23]0101011100102122011217Jiang 2010 [24]1221211210122222211230Wang 2010 [25]1111011221012102011221Xiong 2011 [26]1121111120100022012221Wang 2011 [27]1111111110011121011219Tian 2012 [28]1221211221000102011020Duan 2013 [29]1111011221110122011020Chen 2013 [30]1111211201110122011222Wang 2013 [31]1100002220000222001015Xu 2014 [32]1111012221101122111022Yu 2014 [33]1211012221012222011024Shen 2015 [34]1111012221110122011021Liu 2015 [35]0111022200112112012222Liu 2015 [36]1112011201112121011222Zeng 2015 [37]1212011201012101021019Tian 2015 [38]1111011221111122111022Sun 2015 [39]1111012221111122211024Zhang 2015 [40]1111012201010222011019Fu 2015 [41]1121011222022122011226Chang Lee 2016 [42]1121112121100022011222Chen 2016 [43]1121012221112122021227Tan 2016 [44]1120211100002022011219Wang 2016 [45]1111011221000121011017Wang 2016 [46]1111012221012122011224Wang 2016 [47]1101012211110122011019Xin 2016 [48]0111001200011001011011Wang 2017 [49]0111011221112102011221Zhang 2017 [50]1110011111010122011218Guo 2017 [51]1111012201110022011220Xin 2017 [52]1222212210002122011024Xin 2017 [53]1100011110010122000214Guo 2018 [54]1111211211111122011224Wang 2018 [55]1111012210112222012225Wang 2018 [56]1222111121002122011225Wang 2018 [57]1121011211110122011222Wang 2018 [58]1121112210112202012225Deng 2018 [59]1101012221002212011222Huang 2018 [60]1121211221102222011227Ma 2018 [61]1111011201110022001218Dong 2018 [62]1111012101112022011221Zhao 2018 [63]1111212221100022011223Zheng 2018 [64]1111011221100022011220Li 2018 [65]1112111221000122011020Luo 2018 [66]1121211221002222011226Cui 2019 [67]1111011221100122011019Luo 2019 [9]0121212221112222011228Wang 2019 [68]1221212220110102011022Wang 2019 [69]1111211221122022021227Ma 2019 [70]1221111121102022011224Ji 2019 [71]1121211201110022011222Zheng 2019 [72]1111012201110122011221Ning 2019 [73]1111011200102222111222Sun 2019 [74]1111011201112101011219Cui 2020 [75]1111011201120122011221Hao 2020 [76]1111212221110122011225Fang 2020 [77]1101012201110122011220Sun 2020 [78]1121211201100122011222Sun 2020 [79]1121111201110122011222Yang 2020 [80]1111012101102022011220Category score (quality obtained)6077786838668611769514049616411412886471941403Maximum score (quality expected)67134134134134134134134134134671341341341341341341341341342546Ratio: quality score/maximum score0.900.570.580.510.290.490.640.870.510.380.600.370.460.480.850.960.060.480.530.700.551061: title; 2: abstract; 3: introduction-background; 4: introduction-objectives; 5: methods-ethical statement; 6: study design; 7: experimental procedure; 8: experimental animals; 9: housing and husbandry; 10: sample size; 11: allocation; 12: experimental outcomes; 13: statistics; 14: results-baseline data; 15: number analysed; 16: outcome and estimation; 17: adverse events; 18: discussion-interpretation/scientific implications; 19: general applicability/relevance; 20: funding; Total: represents total score obtained by each study out of a maximum of 38 points.


### 3.3. Risk of Bias

Risk of bias was appraised for each included study. 37 studies were thought to have low risk of sequence generation. 23 studies were judged as having low risk of baseline characteristics. No study was considered to have low risk of allocation concealment, random housing, blinding against performance bias, random outcome assessment, and blinding against detection bias. 56 studies were thought to have low risk of incomplete data. All of the 67 studies were thought to have low risk of selective outcome reporting and other bias. Thus, all studies were judged as having high risk of bias. Details of bias assessment information of identified studies are shown in Table3.Table 3
Risk of bias assessment according to the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool.
StudiesSYRCLE items12345678910Chen 2001 [15]?nnnnnnyyyChen 2002 [16]?nnnnnnyyyLiu 2003 [17]?ynnnnn?yySu 2003 [18]??nnnnnyyyYang 2006 [19]?ynnnnnyyyYang 2008 [20]?nnnnnnyyyWei 2008 [21]??nnnnnyyyQian 2008 [22]??nnnnnyyyLiu 2009 [23]y?nnnnnyyyJiang 2010 [24]?ynnnnnyyyWang 2010 [25]?ynnnnn?yyXiong 2011 [26]??nnnnnyyyWang 2011 [27]?y?nnnnyyyTian 2012 [28]?nnnnnn?yyDuan 2013 [29]y?nnnnnyyyChen 2013 [30]y?nnnnnyyyWang 2013 [31]??nnnnnyyyXu 2014 [32]y?nnnnnyyyYu 2014 [33]?ynnnnnyyyShen 2015 [34]yynnnnnyyyLiu 2015 [35]yynnnnnnyyLiu 2015 [36]yynnnnnyyyZeng 2015 [37]?ynnnnn?yyTian 2015 [38]y?nnnnnyyySun 2015 [39]y?nnnnnyyyZhang 2015 [40]?ynnnnnyyyFu 2015 [41]?ynnnnnyyyChang Lee 2016 [42]??nnnnnyyyChen 2016 [43]??nnnnnyyyTan 2016 [44]?nnnnnnyyyWang 2016 [45]?ynnnnnyyyWang 2016 [46]?ynnnnnyyyWang 2016 [47]y?nnnnnyyyXin 2016 [48]nynnnnnnyyWang 2017 [49]y?nnnnn?yyZhang 2017 [50]??nnnnnyyyGuo 2017 [51]ynnnnnnyyyXin 2017 [52]n?nnnnnyyyXin 2017 [53]?ynnnnnyyyGuo 2018 [54]y?nnnnnyyyWang 2018 [55]y?nnnnnyyyWang 2018 [56]??nnnnnyyyWang 2018 [57]yynnnnnyyyWang 2018 [58]ynnnnn??yyDeng 2018 [59]?ynnnnn?yyHuang 2018 [60]yynnnnnyyyMa 2018 [61]ynnnnnnyyyDong 2018 [62]ynnnnnnyyyZhao 2018 [63]ynnnnnnyyyZheng 2018 [64]y?nnnnnyyyLi 2018 [65]??nnnnnyyyLuo 2018 [66]?ynnnnnyyyCui 2019 [67]y?nnnnnyyyLuo 2019 [9]yynnnnnyyyWang 2019 [68]y?nnnnn?yyWang 2019 [69]y?nnnnnyyyMa 2019 [70]y?nnnnnyyyJi 2019 [71]ynnnnnnyyyZheng 2019 [72]y?nnnnnyyyNing 2019 [73]yynnnnnyyySun 2019 [74]yynnnnn?yyCui 2020 [75]y?nnnnnyyyHao 2020 [76]y?nnnnnyyyFang 2020 [77]y?nnnnnyyySun 2020 [78]y?nnnnnyyySun 2020 [79]y?nnnnnyyyYang 2020 [80]ynnnnnnyyy1: sequence generation; 2: baseline characteristics; 3: allocation concealment; 4: random housing; 5: blinding of participants and personnel; 6: random outcome assessment; 7: blinding of outcome assessment; 8: incomplete outcome data; 9: selective outcome reporting; 10: other bias; y: low risk of bias; ?: unclear; n: high risk of bias.


### 3.4. Effectiveness

#### 3.4.1. SBP

61 studies including 1385 animals conducted the meta-analysis of SBP value. Compared with the hypertension model group, acupuncture showed a mean reduction of SBP for 25.37 mmHg with significant heterogeneity in a pooled analysis of 60 studies (Figure2) (MD −25.37, 95% CI: −29.18 to −21.56, P<0.0001; heterogeneity: chi2 = 2746.83, df = 59 (P<0.0001), I2 = 97.6%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction of SBP for 21.47 mmHg with statistically significant heterogeneity in a pooled analysis of 19 studies (Figure 3) (MD −21.47, 95% CI: −27.04 to −15.89, P<0.0001; heterogeneity: chi2 = 342.60, df = 18 (P<0.0001), I2 = 94.7%).Figure 2
SBP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 3
SBP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


#### 3.4.2. DBP

23 studies including 523 animals conducted the meta-analysis of DBP value. Compared with the hypertension model group, acupuncture showed a mean reduction of DBP for 21.26 mmHg with statistically significant heterogeneity in a pooled analysis of 22 studies (Figure4) (MD −21.26, 95% CI: −26.21 to −16.30, P<0.0001; heterogeneity: chi2 = 425.69, df = 21 (P<0.0001), I2 = 95.1%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction in DBP for 19.11 mmHg with statistically significant heterogeneity in a pooled analysis of 7 studies (Figure 5) (MD −19.11, 95% CI: −27.89 to −10.33, P<0.0001; heterogeneity: chi2 = 58.32, df = 6 (P<0.0001), I2 = 89.7%).Figure 4
DBP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 5
DBP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


#### 3.4.3. MAP

12 studies including 274 animals conducted the meta-analysis of MAP value. Compared with the hypertension model group, acupuncture showed a mean reduction of MAP for 21.96 mmHg with statistically significant heterogeneity in a pooled analysis of 12 studies (Figure6) (MD −21.96, 95% CI: −29.31 to −14.62, P<0.001; heterogeneity: chi2 = 284.15, df = 11 (P<0.0001), I2 = 96.1%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction in MAP for 16.72 mmHg with statistically significant heterogeneity in a pooled analysis of 5 studies (Figure 7) (MD −16.72, 95% CI: −23.98 to −9.46, P<0.0001; heterogeneity: chi2 = 51.97, df = 4 (P<0.0001), I2 = 92.3%).Figure 6
MAP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 7
MAP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


### 3.5. 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 TablesS1–S5. The 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 (FiguresS1–S6).


### 3.6. Assessment of Publication Bias

Funnel plot of all outcome measures between different groups (Figures8–13) showed symmetry, indicating no significant publication bias. Egger’s test of different comparisons of SBP/DBP/MAP showed no publication bias present. Details of Egger’s test are shown in Table S6.Figure 8
Funnel plot of SBP: acupuncture vs. hypertension.Figure 9
Funnel plot of SBP: acupuncture vs. sham-acupuncture.Figure 10
Funnel plot of DBP: acupuncture vs. hypertension.Figure 11
Funnel plot of DBP: acupuncture vs. sham-acupuncture.Figure 12
Funnel plot of MAP: acupuncture vs. hypertension.Figure 13
Funnel plot of MAP: acupuncture vs. sham-acupuncture.


### 3.7. Related Mechanisms

The underlying molecular mechanisms of acupuncture involved in the present review for hypertension are summarized as follows: (1) inhibition of inflammatory factors including tumor necrosis factor-α (TNF-α) [21], interleukin-6, and C-reactive protein [40, 46], as well as inflammation-related genes [63], through toll-like receptor 4 [66] in the PVN; (2) reduction of oxidative stress reactions [64, 72–74, 79] by inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by downregulation of p38 mitogen-activated protein kinases and extracellular signal-regulated protein kinase 1/2 [58]; (3) inhibition of AngII activity in the plasma [23, 32, 62, 67, 75], myocardium [59, 71], thoracic aorta [69], rostral ventrolateral medulla [62], kidney [62], etc. and reversion of the artery remodeling [53] and left ventricle [45, 57] through mitogen-activated protein kinase signaling [24]; (4) inhibition of glucocorticoid system, including corticotropin-releasing hormone, adrenocorticotropic hormone, and cortisol and glucocorticoids receptor [48, 52]; and (5) mediation of the balance between vasorelaxation factors and vasomotor factors and improvement of the endothelial dysfunction [15, 16, 21, 25–27, 34, 39, 49, 57, 59]. This is consistent with the genomic study of acupuncture, which suggested acupuncture may regulate multiple biological processes mainly involving oxidative stress, inflammation, and vascular endothelial function [70].


## 3.1. Study Characteristics

Totally, 1522 animals were included for comparison. Of the hypertension models in these studies, 63 used SHR, 1 used AngII infusion [44], and 3 used renal artery stenosis [17, 21, 22]. 3 studies simultaneously assessed SBP/DBP/MAP as the outcome measures [42, 48, 52]. 20 studies assessed SBP and DBP as the outcome measures [23, 32, 35, 36, 38, 41, 43, 45, 47, 49, 50, 53, 55, 56, 60, 62, 68, 72, 79, 80]. 3 studies assessed SBP and MAP as the outcome measures [37, 58, 70]. 35 studies assessed only SBP as the outcome measures [9, 17–22, 24–27, 29–31, 33, 34, 39, 40, 46, 51, 54, 57, 59, 61, 63–65, 67, 69, 71, 73, 75–78]. 6 studies assessed only MAP as the outcome measure [15, 16, 28, 44, 66, 74]. 40 studies adopted manual acupuncture [9, 17, 20, 25, 29, 32–41, 43, 45–47, 50, 51, 53, 54, 57–63, 65–68, 70, 71, 75–78], of which 35 adopted manipulation instead of simple needle penetration [9, 17, 20, 25, 29, 32–41, 43, 45–47, 50, 51, 53, 54, 57, 59–61, 63, 65, 66, 68, 71, 76–78]. 20 studies used EA as intervention [15, 16, 18, 21–23, 27, 28, 30, 31, 42, 44, 48, 52, 64, 69, 72–74, 79]. 4 studies combined manipulation and EA as intervention [24, 49, 55, 56]. 2 studies used other auxiliary devices combined with acupuncture as treatment [19, 80]. The needle retaining time ranged from 0.5 minute to 30 minutes. The duration of acupuncture ranged from 1 time to 48 times. Animal age of treatment initiation ranged from 8 weeks to 34 weeks. Study characteristics are shown in Table 1.Table 1
Characteristics of included studies.
StudyAnimals (species, sex, weight)ModelControl groupOutcome measurementWeek-age of acupuncture initiation (weeks)Week-age of BP measurement (weeks)Acupuncture methodsAcupointsAcupuncture retaining time (minutes)Acupuncture duration (sessions)Chen 2001 [15]SHR, male and female, 189.17 ± 31.26 gSHRHTNMAP1013EAST361512Chen 2002 [16]SHR, male and female, NRSHRHTNMAP1114EAST361515Liu 2003 [17]Wistar, NR, 200 ± 20 gRenal ischemiaHTNSBP2224ManipST36, SP3, PC6, KI12014Su 2003 [18]SHR, male, NRSHRHTNSBP922EAST36, LI113045Yang 2006 [19]SHR, male, 270 ± 10 gSHRHTNSBP1215Other deviceST36, LR3315Yang 2008 [20]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBPNRNRManipLR357Wei 2008 [21]SD, male and female, 190 ± 10 gRenal ischemiaHTNSBPNRNREALI112028Qian 2008 [22]SD, male and female, 175 ± 25 gRenal ischemiaHTNSBPNRNREALI112056Liu 2009 [23]SHR, male, 220 ± 20 gSHRHTN, Sham-acuSBP, DBPNRNREAHT7301Jiang 2010 [24]SHR, male, 150 ± 50 gSHRHTNSBP3437EALI11, GB20, SP61518Wang 2010 [25]SHR, male, 200 ± 20 gSHRHTNSBP910ManipLR357Xiong 2011 [26]SHR, male, NRSHRHTNSBP1321EAST36NR40Wang 2011 [27]SHR, male, 200 ± 20 gSHRHTNSBPNRNREALR357Tian 2012 [28]SHR, male, 220 ± 30SHRHTNMAP816EAST36, DU202028Duan 2013 [29]SHR, male, 200 ± 20 gSHRHTNSBP1113ManipLR32014Chen 2013 [30]SHR, male, 385.22 ± 21.26SHRHTNSBP2430EAST36, LI112042Wang 2013 [31]SHR, male, 360 ± 20 gSHRHTNSBP2632EAST36, LI112042Xu 2014 [32]SHR, male, 260 ± 20SHRHTNSBP, DBP1216ManipLR31024Yu 2014 [33]SHR, male, 210 ± 10SHRHTN, Sham-acuSBP1213ManipLR3, KI357Shen 2015 [34]SHR, male, 245 ± 25SHRHTNSBP1721ManipST9120Liu 2015 [35]SHR, male, 215 ± 25SHRHTNSBP, DBP1721ManipST9120Liu 2015 [36]SHR, male, 215 ± 25SHRHTNSBP, DBP1721ManipST9120Zeng 2015 [37]SHR, male, 255 ± 5SHRHTNSBP, MAP1315ManipLR30.512Tian 2015 [38]SHR, male, 225 ± 25SHRHTNSBP, DBP1216ManipLR31024Sun 2015 [39]SHR, male, 225 ± 25SHRHTNSBP1216ManipLR31024Zhang 2015 [40]SHR, male, 210 ± 10SHRHTN, Sham-acuSBP1213ManipKI357Fu 2015 [41]SHR, male, 300 ± 10 gSHRSham-acuSBP, DBP1822ManipST9128Chang Lee 2016 [42]SHR, male, NRSHRHTN, Sham-acuSBPNRNREALR33021Chen 2016 [43]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBP, DBP1011ManipLR357Tan 2016 [44]SD, male, 190 ± 10 gAngII-inducedHTNMAPNRNREALI112014Wang 2016 [45]SHR, male, 250 ± 20 gSHRHTN, Sham-acuSBP, DBP1114ManipST9221Wang 2016 [46]SHR, male, 210 ± 10SHRHTN, Sham-acuSBP1213ManipKI357Wang 2016 [47]SHR, male, 250 ± 20SHRHTNSBP, DBP1115ManipST36, LR32028Xin 2016 [48]SHR, male, 255 ± 15SHRHTNSBP, DBP, MAP1220EAPC63056Wang 2017 [49]SHR, male, 260 ± 9.04SHRHTNSBP, DBP, MAP1216Manip + EAST36, LI111620Zhang 2017 [50]SHR, male, 190 ± 10 gSHRHTNSBP, DBPNRNRManipLR3, PC63015Guo 2017 [51]SHR, male, 190 ± 10 gSHRHTNSBP913ManipST92028Xin 2017 [52]SHR, male, 255 ± 15 gSHRHTNSBP, DBP, MAP1321EAPC63056Xin 2017 [53]SHR, male and female, 220 ± 20 gSHRHTNSBP, DBPNRNRManipST36, LI113021Guo 2018 [54]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBP1010ManipST9301Wang 2018 [55]SHR, male, 259.17 ± 9.71SHRHTNSBP, DBP1216Manip + EAST36, LI111520Wang 2018 [56]SHR, male, NRSHRHTN, Sham-acuSBP, MAP1214MAST36, LR31514Wang 2018 [57]SHR, male, 225 ± 25 gSHRHTNSBP913ManipLR32024Wang 2018 [58]SHR, male, 259.17 ± 9.71SHRHTNSBP, DBP1216Manip + EAST36, LI111520Deng 2018 [59]SHR, male, 190 ± 10 gSHRHTN, Sham-acuSBP1216ManipKI3524Huang 2018 [60]SHR, male, 235 ± 15 gSHRHTN, Sham-acuSBP, DBP1115ManipLR3, KI31524Ma 2018 [61]SHR, male, 190 ± 10 gSHRHTNSBP913ManipST92024Dong 2018 [62]SHR, male, 295 ± 15SHRHTNSBP, DBP1725MAST36, ST9, LI112048Zhao 2018 [63]SHR, male, 190 ± 10 gSHRHTNSBP1014ManipST92028Zheng 2018 [64]SHR, male, 275 ± 25 gSHRHTNSBP1721ManipST92028Li 2018 [65]SHR, male, 240 ± 10SHRHTN, Sham-acuSBP1216ManipST36, PC6, BL15, BL203028Luo 2018 [66]SHR, male, 190 ± 10 gSHRHTN, Sham-acuMAP1216EAST36, DU202028Cui 2019 [67]SHR, male, 240 ± 20 gSHRHTNSBP10-1118-19MAST36, ST7, LI113040Luo 2019 [9]SHR, male, 230 ± 20 gSHRHTN, Sham-acuSBP1216ManipLR3524Wang 2019 [68]SHR, male, 235 ± 15 gSHRHTNSBP1216EADU20, LR32030Wang 2019 [69]SHR, male, 280 ± 8 gSHRHTN, Sham-acuSBP, DBP1314ManipLR3107Ma 2019 [70]SHR, male, NRSHRHTNSBP, MAP1214MALR32014Ji 2019 [71]SHR, male, 215 ± 15 gSHRHTNSBP1115ManipDU20, LR32028Zheng 2019 [72]SHR, male, 275 ± 25 gSHRHTNSBP, DBP1721EAST72028Ning 2019 [73]SHR, male, 300 ± 30 gSHRHTNSBP1628EABL231542Sun 2019 [74]SHR, male, 275 ± 25 gSHRHTNMAP1721EAST72028Cui 2020 [75]SHR, male, 180 ± 20 gSHRHTNSBP1014MAST36, ST7, LI113020Hao 2020 [76]SHR, male, 220 ± 30 gSHRHTNSBP1012ManipLR32014Fang 2020 [77]SHR, male, 200 ± 20 gSHRHTNSBP913ManipST36, LI4, LR3528Sun 2020 [78]SHR, male, 220 ± 30 gSHRHTNSBP1012ManipLR32015Sun 2020 [79]SHR, male, 275 ± 25 gSHRHTNSBP, DBP1721EAST72028Yang 2020 [80]SHR, male, NRSHRHTNSBP, DBP1620OtherLR3120NR: not reported; SHR: spontaneously hypertension rat; HTN: hypertension; Sham-acu: sham-acupuncture; SBP: systolic blood pressure; DBP: diastolic blood pressure; MAP: mean arterial pressure; EA: electroacupuncture; MA: manual acupuncture; Manip: manipulation.


## 3.2. Quality Assessment

Twenty items were evaluated according to the ARRIVE guidelines. According to ratio quality score/maximum score, the items 5/6/10/12/13/14/17/18 were considered to be poor. The items 2/3/4/7/9/11/19/20 were considered to be average. Also, the items 1/8/15/16 were considered to be excellent. Detailed information of identified studies’ quality is shown in Table2.Table 2
Quality assessment according to the Animal Research: Reporting In Vivo Experiment (ARRIVE) guidelines.
StudiesARRIVE items1234567891011121314151617181920TotalChen 2001 [15]0111010200010022011215Chen 2002 [16]1111010100010022001214Liu 2003 [17]0111010101010102011012Su 2003 [18]1111010100000122001214Yang 2006 [19]1111011201011122012019Yang 2008 [20]1211011101012222011222Wei 2008 [21]1201011111010122111018Qian 2008 [22]1101010101010122001215Liu 2009 [23]0101011100102122011217Jiang 2010 [24]1221211210122222211230Wang 2010 [25]1111011221012102011221Xiong 2011 [26]1121111120100022012221Wang 2011 [27]1111111110011121011219Tian 2012 [28]1221211221000102011020Duan 2013 [29]1111011221110122011020Chen 2013 [30]1111211201110122011222Wang 2013 [31]1100002220000222001015Xu 2014 [32]1111012221101122111022Yu 2014 [33]1211012221012222011024Shen 2015 [34]1111012221110122011021Liu 2015 [35]0111022200112112012222Liu 2015 [36]1112011201112121011222Zeng 2015 [37]1212011201012101021019Tian 2015 [38]1111011221111122111022Sun 2015 [39]1111012221111122211024Zhang 2015 [40]1111012201010222011019Fu 2015 [41]1121011222022122011226Chang Lee 2016 [42]1121112121100022011222Chen 2016 [43]1121012221112122021227Tan 2016 [44]1120211100002022011219Wang 2016 [45]1111011221000121011017Wang 2016 [46]1111012221012122011224Wang 2016 [47]1101012211110122011019Xin 2016 [48]0111001200011001011011Wang 2017 [49]0111011221112102011221Zhang 2017 [50]1110011111010122011218Guo 2017 [51]1111012201110022011220Xin 2017 [52]1222212210002122011024Xin 2017 [53]1100011110010122000214Guo 2018 [54]1111211211111122011224Wang 2018 [55]1111012210112222012225Wang 2018 [56]1222111121002122011225Wang 2018 [57]1121011211110122011222Wang 2018 [58]1121112210112202012225Deng 2018 [59]1101012221002212011222Huang 2018 [60]1121211221102222011227Ma 2018 [61]1111011201110022001218Dong 2018 [62]1111012101112022011221Zhao 2018 [63]1111212221100022011223Zheng 2018 [64]1111011221100022011220Li 2018 [65]1112111221000122011020Luo 2018 [66]1121211221002222011226Cui 2019 [67]1111011221100122011019Luo 2019 [9]0121212221112222011228Wang 2019 [68]1221212220110102011022Wang 2019 [69]1111211221122022021227Ma 2019 [70]1221111121102022011224Ji 2019 [71]1121211201110022011222Zheng 2019 [72]1111012201110122011221Ning 2019 [73]1111011200102222111222Sun 2019 [74]1111011201112101011219Cui 2020 [75]1111011201120122011221Hao 2020 [76]1111212221110122011225Fang 2020 [77]1101012201110122011220Sun 2020 [78]1121211201100122011222Sun 2020 [79]1121111201110122011222Yang 2020 [80]1111012101102022011220Category score (quality obtained)6077786838668611769514049616411412886471941403Maximum score (quality expected)67134134134134134134134134134671341341341341341341341341342546Ratio: quality score/maximum score0.900.570.580.510.290.490.640.870.510.380.600.370.460.480.850.960.060.480.530.700.551061: title; 2: abstract; 3: introduction-background; 4: introduction-objectives; 5: methods-ethical statement; 6: study design; 7: experimental procedure; 8: experimental animals; 9: housing and husbandry; 10: sample size; 11: allocation; 12: experimental outcomes; 13: statistics; 14: results-baseline data; 15: number analysed; 16: outcome and estimation; 17: adverse events; 18: discussion-interpretation/scientific implications; 19: general applicability/relevance; 20: funding; Total: represents total score obtained by each study out of a maximum of 38 points.


## 3.3. Risk of Bias

Risk of bias was appraised for each included study. 37 studies were thought to have low risk of sequence generation. 23 studies were judged as having low risk of baseline characteristics. No study was considered to have low risk of allocation concealment, random housing, blinding against performance bias, random outcome assessment, and blinding against detection bias. 56 studies were thought to have low risk of incomplete data. All of the 67 studies were thought to have low risk of selective outcome reporting and other bias. Thus, all studies were judged as having high risk of bias. Details of bias assessment information of identified studies are shown in Table3.Table 3
Risk of bias assessment according to the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool.
StudiesSYRCLE items12345678910Chen 2001 [15]?nnnnnnyyyChen 2002 [16]?nnnnnnyyyLiu 2003 [17]?ynnnnn?yySu 2003 [18]??nnnnnyyyYang 2006 [19]?ynnnnnyyyYang 2008 [20]?nnnnnnyyyWei 2008 [21]??nnnnnyyyQian 2008 [22]??nnnnnyyyLiu 2009 [23]y?nnnnnyyyJiang 2010 [24]?ynnnnnyyyWang 2010 [25]?ynnnnn?yyXiong 2011 [26]??nnnnnyyyWang 2011 [27]?y?nnnnyyyTian 2012 [28]?nnnnnn?yyDuan 2013 [29]y?nnnnnyyyChen 2013 [30]y?nnnnnyyyWang 2013 [31]??nnnnnyyyXu 2014 [32]y?nnnnnyyyYu 2014 [33]?ynnnnnyyyShen 2015 [34]yynnnnnyyyLiu 2015 [35]yynnnnnnyyLiu 2015 [36]yynnnnnyyyZeng 2015 [37]?ynnnnn?yyTian 2015 [38]y?nnnnnyyySun 2015 [39]y?nnnnnyyyZhang 2015 [40]?ynnnnnyyyFu 2015 [41]?ynnnnnyyyChang Lee 2016 [42]??nnnnnyyyChen 2016 [43]??nnnnnyyyTan 2016 [44]?nnnnnnyyyWang 2016 [45]?ynnnnnyyyWang 2016 [46]?ynnnnnyyyWang 2016 [47]y?nnnnnyyyXin 2016 [48]nynnnnnnyyWang 2017 [49]y?nnnnn?yyZhang 2017 [50]??nnnnnyyyGuo 2017 [51]ynnnnnnyyyXin 2017 [52]n?nnnnnyyyXin 2017 [53]?ynnnnnyyyGuo 2018 [54]y?nnnnnyyyWang 2018 [55]y?nnnnnyyyWang 2018 [56]??nnnnnyyyWang 2018 [57]yynnnnnyyyWang 2018 [58]ynnnnn??yyDeng 2018 [59]?ynnnnn?yyHuang 2018 [60]yynnnnnyyyMa 2018 [61]ynnnnnnyyyDong 2018 [62]ynnnnnnyyyZhao 2018 [63]ynnnnnnyyyZheng 2018 [64]y?nnnnnyyyLi 2018 [65]??nnnnnyyyLuo 2018 [66]?ynnnnnyyyCui 2019 [67]y?nnnnnyyyLuo 2019 [9]yynnnnnyyyWang 2019 [68]y?nnnnn?yyWang 2019 [69]y?nnnnnyyyMa 2019 [70]y?nnnnnyyyJi 2019 [71]ynnnnnnyyyZheng 2019 [72]y?nnnnnyyyNing 2019 [73]yynnnnnyyySun 2019 [74]yynnnnn?yyCui 2020 [75]y?nnnnnyyyHao 2020 [76]y?nnnnnyyyFang 2020 [77]y?nnnnnyyySun 2020 [78]y?nnnnnyyySun 2020 [79]y?nnnnnyyyYang 2020 [80]ynnnnnnyyy1: sequence generation; 2: baseline characteristics; 3: allocation concealment; 4: random housing; 5: blinding of participants and personnel; 6: random outcome assessment; 7: blinding of outcome assessment; 8: incomplete outcome data; 9: selective outcome reporting; 10: other bias; y: low risk of bias; ?: unclear; n: high risk of bias.


## 3.4. Effectiveness

### 3.4.1. SBP

61 studies including 1385 animals conducted the meta-analysis of SBP value. Compared with the hypertension model group, acupuncture showed a mean reduction of SBP for 25.37 mmHg with significant heterogeneity in a pooled analysis of 60 studies (Figure2) (MD −25.37, 95% CI: −29.18 to −21.56, P<0.0001; heterogeneity: chi2 = 2746.83, df = 59 (P<0.0001), I2 = 97.6%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction of SBP for 21.47 mmHg with statistically significant heterogeneity in a pooled analysis of 19 studies (Figure 3) (MD −21.47, 95% CI: −27.04 to −15.89, P<0.0001; heterogeneity: chi2 = 342.60, df = 18 (P<0.0001), I2 = 94.7%).Figure 2
SBP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 3
SBP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


### 3.4.2. DBP

23 studies including 523 animals conducted the meta-analysis of DBP value. Compared with the hypertension model group, acupuncture showed a mean reduction of DBP for 21.26 mmHg with statistically significant heterogeneity in a pooled analysis of 22 studies (Figure4) (MD −21.26, 95% CI: −26.21 to −16.30, P<0.0001; heterogeneity: chi2 = 425.69, df = 21 (P<0.0001), I2 = 95.1%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction in DBP for 19.11 mmHg with statistically significant heterogeneity in a pooled analysis of 7 studies (Figure 5) (MD −19.11, 95% CI: −27.89 to −10.33, P<0.0001; heterogeneity: chi2 = 58.32, df = 6 (P<0.0001), I2 = 89.7%).Figure 4
DBP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 5
DBP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


### 3.4.3. MAP

12 studies including 274 animals conducted the meta-analysis of MAP value. Compared with the hypertension model group, acupuncture showed a mean reduction of MAP for 21.96 mmHg with statistically significant heterogeneity in a pooled analysis of 12 studies (Figure6) (MD −21.96, 95% CI: −29.31 to −14.62, P<0.001; heterogeneity: chi2 = 284.15, df = 11 (P<0.0001), I2 = 96.1%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction in MAP for 16.72 mmHg with statistically significant heterogeneity in a pooled analysis of 5 studies (Figure 7) (MD −16.72, 95% CI: −23.98 to −9.46, P<0.0001; heterogeneity: chi2 = 51.97, df = 4 (P<0.0001), I2 = 92.3%).Figure 6
MAP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 7
MAP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


## 3.4.1. SBP

61 studies including 1385 animals conducted the meta-analysis of SBP value. Compared with the hypertension model group, acupuncture showed a mean reduction of SBP for 25.37 mmHg with significant heterogeneity in a pooled analysis of 60 studies (Figure2) (MD −25.37, 95% CI: −29.18 to −21.56, P<0.0001; heterogeneity: chi2 = 2746.83, df = 59 (P<0.0001), I2 = 97.6%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction of SBP for 21.47 mmHg with statistically significant heterogeneity in a pooled analysis of 19 studies (Figure 3) (MD −21.47, 95% CI: −27.04 to −15.89, P<0.0001; heterogeneity: chi2 = 342.60, df = 18 (P<0.0001), I2 = 94.7%).Figure 2
SBP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 3
SBP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


## 3.4.2. DBP

23 studies including 523 animals conducted the meta-analysis of DBP value. Compared with the hypertension model group, acupuncture showed a mean reduction of DBP for 21.26 mmHg with statistically significant heterogeneity in a pooled analysis of 22 studies (Figure4) (MD −21.26, 95% CI: −26.21 to −16.30, P<0.0001; heterogeneity: chi2 = 425.69, df = 21 (P<0.0001), I2 = 95.1%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction in DBP for 19.11 mmHg with statistically significant heterogeneity in a pooled analysis of 7 studies (Figure 5) (MD −19.11, 95% CI: −27.89 to −10.33, P<0.0001; heterogeneity: chi2 = 58.32, df = 6 (P<0.0001), I2 = 89.7%).Figure 4
DBP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 5
DBP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


## 3.4.3. MAP

12 studies including 274 animals conducted the meta-analysis of MAP value. Compared with the hypertension model group, acupuncture showed a mean reduction of MAP for 21.96 mmHg with statistically significant heterogeneity in a pooled analysis of 12 studies (Figure6) (MD −21.96, 95% CI: −29.31 to −14.62, P<0.001; heterogeneity: chi2 = 284.15, df = 11 (P<0.0001), I2 = 96.1%). Compared with the sham-acupuncture group, acupuncture showed a mean reduction in MAP for 16.72 mmHg with statistically significant heterogeneity in a pooled analysis of 5 studies (Figure 7) (MD −16.72, 95% CI: −23.98 to −9.46, P<0.0001; heterogeneity: chi2 = 51.97, df = 4 (P<0.0001), I2 = 92.3%).Figure 6
MAP forest plots: acupuncture vs. hypertension. WMD, weighted mean difference.Figure 7
MAP forest plots: acupuncture vs. sham-acupuncture. WMD, weighted mean difference.


## 3.5. 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 TablesS1–S5. The 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 (FiguresS1–S6).


## 3.6. Assessment of Publication Bias

Funnel plot of all outcome measures between different groups (Figures8–13) showed symmetry, indicating no significant publication bias. Egger’s test of different comparisons of SBP/DBP/MAP showed no publication bias present. Details of Egger’s test are shown in Table S6.Figure 8
Funnel plot of SBP: acupuncture vs. hypertension.Figure 9
Funnel plot of SBP: acupuncture vs. sham-acupuncture.Figure 10
Funnel plot of DBP: acupuncture vs. hypertension.Figure 11
Funnel plot of DBP: acupuncture vs. sham-acupuncture.Figure 12
Funnel plot of MAP: acupuncture vs. hypertension.Figure 13
Funnel plot of MAP: acupuncture vs. sham-acupuncture.


## 3.7. Related Mechanisms

The underlying molecular mechanisms of acupuncture involved in the present review for hypertension are summarized as follows: (1) inhibition of inflammatory factors including tumor necrosis factor-α (TNF-α) [21], interleukin-6, and C-reactive protein [40, 46], as well as inflammation-related genes [63], through toll-like receptor 4 [66] in the PVN; (2) reduction of oxidative stress reactions [64, 72–74, 79] by inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by downregulation of p38 mitogen-activated protein kinases and extracellular signal-regulated protein kinase 1/2 [58]; (3) inhibition of AngII activity in the plasma [23, 32, 62, 67, 75], myocardium [59, 71], thoracic aorta [69], rostral ventrolateral medulla [62], kidney [62], etc. and reversion of the artery remodeling [53] and left ventricle [45, 57] through mitogen-activated protein kinase signaling [24]; (4) inhibition of glucocorticoid system, including corticotropin-releasing hormone, adrenocorticotropic hormone, and cortisol and glucocorticoids receptor [48, 52]; and (5) mediation of the balance between vasorelaxation factors and vasomotor factors and improvement of the endothelial dysfunction [15, 16, 21, 25–27, 34, 39, 49, 57, 59]. This is consistent with the genomic study of acupuncture, which suggested acupuncture may regulate multiple biological processes mainly involving oxidative stress, inflammation, and vascular endothelial function [70].


## 4. Discussion

To our knowledge, we present the first systematic review and meta-analysis of the efficacy of acupuncture in treating animal models of hypertension. For the outcome measures, including SBP/DBP/MAP, our meta-analysis showed better results in the acupuncture group than in the model or sham-acupuncture group. This indicates that acupuncture might potentially decrease HBP, though there was high heterogeneity among the results of the analysed studies.


### 4.1. Limitations

Limitations are enumerated as follows. First, being limited in English and Chinese databases, other language studies may be missed. The absence of other language studies may bring about selective bias. Second, negative results were hardly published. Thus, the whole body of positive results might bring about exaggerated efficacy. Third, 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 implied not only differences of laboratory animals but also the acupuncture method details. Though subgroup analyses according to several factors were conducted to explore possible sources of heterogeneity, the results failed to completely account for the heterogeneity. Thus, the present study should be interpreted cautiously.


### 4.2. Efficacy Assessment

Although the effectiveness of acupuncture’s antihypertension in most studies was demonstrated by the value of BP, the ultimate goal of hypertension treatment is to prevent target organ damage and decrease mortality. Among the 67 included studies, 15 assessed the target organ damage, including left ventricle, kidney, and blood vessel remodeling. Limited by study period, none of the studies assessed the mortality of hypertension animals. Thus, target organ damage should be paid attention to in future acupuncture antihypertension studies.Although blood pressure variability (BPV) is not a physiological index daily detected in clinical practice, it 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]. Thus, exploring the effect of acupuncture on hypertension animal BPV may contribute to finding out the potential efficacy of acupuncture.


### 4.3. 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. There 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. The 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. The ARRIVE [89] is a reporting guideline containing a 20-entry checklist about each part of publishing papers. The 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.


## 4.1. Limitations

Limitations are enumerated as follows. First, being limited in English and Chinese databases, other language studies may be missed. The absence of other language studies may bring about selective bias. Second, negative results were hardly published. Thus, the whole body of positive results might bring about exaggerated efficacy. Third, 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 implied not only differences of laboratory animals but also the acupuncture method details. Though subgroup analyses according to several factors were conducted to explore possible sources of heterogeneity, the results failed to completely account for the heterogeneity. Thus, the present study should be interpreted cautiously.


## 4.2. Efficacy Assessment

Although the effectiveness of acupuncture’s antihypertension in most studies was demonstrated by the value of BP, the ultimate goal of hypertension treatment is to prevent target organ damage and decrease mortality. Among the 67 included studies, 15 assessed the target organ damage, including left ventricle, kidney, and blood vessel remodeling. Limited by study period, none of the studies assessed the mortality of hypertension animals. Thus, target organ damage should be paid attention to in future acupuncture antihypertension studies.Although blood pressure variability (BPV) is not a physiological index daily detected in clinical practice, it 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]. Thus, exploring the effect of acupuncture on hypertension animal BPV may contribute to finding out the potential efficacy of acupuncture.


## 4.3. 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. There 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. The 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. The ARRIVE [89] is a reporting guideline containing a 20-entry checklist about each part of publishing papers. The 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.


## 5. 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.



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*Source: 8171636-2021-10-11.xml*