Acupuncture at Zusanli (ST36) for Experimental Sepsis: A Systematic Review

Background Sepsis is a global major health problem with high mortality rates. More effective therapy is needed for treating sepsis. Acupuncture has been used for various diseases, including severe infection, in China for more than 2,000 years. Previous studies reported that acupuncture at Zusanli (ST36) might be effective in treating sepsis, but the efficacy and the quality of evidence remain unclear since there is no systematic review on acupuncture at ST36 for sepsis. Methods Seven databases were searched from the inception of each database up to May 2019. Ultimately, 54 studies using acupuncture at ST36 for the treatment of experimental sepsis were identified in both English and Chinese literature with systematic review procedures. Results Acupuncture might be useful in reducing injuries induced by sepsis in cardiac, lung, kidney, liver, gastrointestinal tract, and immune system. Its potential mechanisms for antisepsis might include reducing oxidative stress and inflammation, improving microcirculatory disturbance, and maintaining the immune balance mediated by dopamine. However, the positive findings should be interpreted with caution due to poor methodological quality and publication bias. Conclusion Acupuncture at ST36 might be a promising complementary strategy for controlling sepsis inflammation, yet further studies are needed.


Introduction
Sepsis is a systemic clinical syndrome induced by inflammatory response from severe infections. e overwhelming inflammatory responses to sepsis may cause multiple organ failure as a result [1][2][3][4], which makes sepsis the leading cause of mortality in noncoronary Intensive Care Units in the world nowadays [5,6]. Although the guidelines of "Surviving Sepsis Campaign" have led to great advances in sepsis management [7] and new antibiotics might be temporarily efficient in helping infection control, the mortality rates are still high and more effective therapy is still urgently needed [8,9].
Acupuncture is one important therapeutic method in traditional Chinese medicine (TCM). It involves the insertion of fine needles in defined points, which is named "acupoints," and usually is followed by stimulation of manual techniques or electrical devices [10]. It has been reported that acupuncture may have a bidirectional regulating effect and antagonize systemic inflammatory response [11][12][13][14]. Zusanli (stomach meridian, ST36) is an acupoint located at 3 cm below the knee joint on the anterior aspect of the leg according to the TCM theory of acupuncture. In the past decade, studies reported the potential of acupuncture at ST36 for infectious diseases due to its numerous effects, such as anti-inflammatory [15], immunoenhancing [16,17], antioxidative [18], accelerating the recovery of various gastrointestinal disorders [19]. Recent studies have further researched acupuncture at ST36 as a treatment for sepsis in humans and animal models [20,21]. However, there is no systematic review of acupuncture at ST36 in treating sepsis; thus systematic assessment of its efficacy and mechanisms is still lacking. Furthermore, systematic reviews of preclinical animal data may help predicting the magnitude and direction of therapeutic effects in human trials [22], identifying potential candidates worthy of further basic research, precluding unnecessary study replication, and contributing to refinement in animal experimentation [23,24]. Herein, we report a systematic review of acupuncture at ST36 in experimental sepsis in this paper with the following objectives: (1) systematically review and evaluate the experimental evidence for acupuncture at ST36, either before or after the onset of sepsis in animal models; (2) determine the efficacy of acupuncture at ST36 in sepsis and explore the impact on the efficacy; (3) analyze the possible mechanisms of acupuncture at ST36 in treating sepsis; (4) propose the refinement for design of future experimental studies and ultimately further clinical trials in human patients in sepsis.

Search Strategy.
We identified studies of acupuncture at ST36 in sepsis animal models from PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL) in e Cochrane Library, EMBASE, Chinese National Knowledge Infrastructure (CNKI), VIP Database, Wanfang Data, and Chinese Biomedical Literature Database (CBM) by using the terms "ST36" OR "zusanli" OR "Tzusanli" OR "Electroacupuncture" OR "Electroacupuncture" OR "EA" OR "acupuncture" OR "acupuncture electric stimulation" OR "AES" AND "sepsis" OR "septicemia" OR "septic shock" OR "endotoxic shock" OR "toxic shock" OR "bloodstream infection" OR "blood stream infection" in English or in Chinese, when appropriate, without language restrictions (search terms for PubMed are listed in the appendix). All databases were searched from the inception up to May 2019. All searches were limited to studies on animals. All included articles and relevant reviews were also hand-searched for the reference lists. Two authors (Rui Chen and Chunmei Yang) identified studies from databases independently. Disagreements were solved after discussion with a third party (Fang Lai).

Eligibility.
Studies must be concerning the effectiveness of acupuncture at ST36 for sepsis, regardless of method of acupuncture (electroacupuncture (EA) or manual acupuncture (MA)), EA waveform parameters, EA current density, EA pulse width, frequency, or course of treatment. To prevent bias, we prespecified the inclusion criteria as follows: (1) studies were included when comparing acupuncture at ST36 as monotherapy or an adjuvant therapy for experimental sepsis and (2) effectiveness was compared with a control group receiving the sham acupuncture therapy, no treatment, positive drug therapy, or the same supporting treatment (such as fluid resuscitation and antibiotics) as the treatment group. Exclusion criteria were preset as follows: (1) studies comparing acupuncture at ST36 as a monotherapy to Chinese herbal medicine (CHM) or acupuncture at another acupoint; (2) duplicate publications; and (3) not focusing on experimental sepsis. Two authors (Guorong Liang and Jiansen Li) screened for included studies independently. Discussions with a third party (Fang Lai) were carried out to solve disagreements.

Data Extraction.
We extracted details from each study as follows: (1) the first author's name and the publication year; (2) model of sepsis, model induction method details, anesthetized method, number of animals, animal species, sex, age, weight, comorbidities (such as hypertension, diabetes, and aged), and study design; (3) method of acupuncture, waveform parameters, current density, frequency, pulse width, course of treatment, intervention of control group, and supportive care for animals; and (4) outcome measures, outcomes assessments time, and intergroup differences.
If the data were missing or conflicting, we tried to contact the authors to get additional information. When no further information is available and critical data remain missing or conflicting, the study would be excluded. Two authors (Xuelian Yin and Caixia Tan) extracted data independently. Discussions were carried out with a third party (Fang Lai) to solve any disagreements.

Study
Selection. 4417 papers were retrieved from the seven above-mentioned databases. 4179 papers were excluded with at least one of the following reasons: (1) being a case report or review; (2) not being an animal research; and (3) not focusing on sepsis. 238 articles remained after going through the titles and abstracts, of which 164 records were removed for being duplicates. Another 1 paper was excluded for no full-text article was available with effort. By full-text reviewing, 16 studies were excluded according to the exclusion criteria, and another 3 articles were excluded for the contradiction between the method and the result. Ultimately, 54 eligible studies were identified. Summary of the study selection process was listed in a flow diagram (Figure 1

Experimental Animals.
e animal species included Sprague-Dawley (SD) rats, Wistar rats (including 3 articles [54,55]which reported as "Wastar rats" by mistake), Wildtype C57BL/6J mice, and New Zealand white rabbits (including 3 articles [32,39,46] reported as "New England rabbits" by mistake). 96.3% of the trials utilized male animals (52 out of the 54 trials), while only 1 trial [62] used both male and female animals and 1 study [49] did not report the sex of the animals.

Sepsis
One study [21] utilized both LPS injection model and CLP model in different episodes, while 3 studies reported utilizing the 20-35% of the total body surface area (TBSA) III degree burned on the back of the rat by boiling water (12-15 s), following by a 5 mg/kg Muramyl dipeptide IV injection [67,70,71].
Details about sepsis models of the included studies are summarized in Table 1.

Sample
Sizes. 50 articles reported the design number of animal in the treatment group (TG) for each outcome index at each time phase, which varied from 3 to 20, while 4 articles [32,41,49,69] did not report the design number of animals in each subgroup. 44 articles reported the actual numbers of animal samples of different groups in Results, 1 article [30] partially reported, while 9 articles [32, 51-53, 58, 59, 68, 78, 81] had no report on numbers of samples in Results.
Song et al., [70] 20% of the TBSA III degree burns on the back of the rat by boiling water (99°C-100°C, 12 s), following by 5 mg/kg MDP i.v. Villegas-Bastida et al., [69] CLP, ligated just proximal to the ileocecal valve, perforated twice with 21-gauge needles at 5 mm distal to the point of ligation Wang et al., [49] 30 mg/kg LPS, i.v. Wang et al., [64] CLP, ligated the root of the cecum, perforated 3 times with a no. 16 needle Wang et al., [78] CLP, ligated 1/3 of the cecum from the ileocecal valve with no. 0-4 silk, perforated with 18-gauge needles Wu et al., [66] CLP, ligated the root of the cecum, perforated 4 times with a no. 16 needle Wu et al., [81] CLP, ligated the root of the cecum, perforated 4 times with a no. 16 needle Wu et al., [76] CLP, ligated the root of the cecum, perforated 4 times with a size 16 needle Xu et al., [65] CLP, no details Yang et al., [50] CLP, ligated the root of the cecum, perforated 2 pores with a no. 12 needle Zhang et al., [59] CLP, ligated the root of the cecum, perforated 3 times with a size 16 needle Zhang et al., [71] 20% of the TBSA III degree burns on the back of the rat by boiling water (99°C-100°C, 12 s), following 5 mg/kg MDP i.v. Zhang et al., [79] CLP, ligated the root of the cecum, perforated 3 times with no. 16 needle Zhang et al., [80] CLP, ligated the lower section of the cecum (25% from the blind side of its total length) using 4-0 silk threads, perforated once with a 19-gauge needle Zhu et al., [73] CLP, ligated the base of the cecum, below the ileocecal valve, with a 2.0-silk, perforated 2 times with no.18 needle Torres-Rosas et al., [21] (1) 6 mg/kg LPS, intraperitoneally

Quality of Studies and Publication
Bias. Study quality checklist (SQC) scores varied from 2 to 10 out of a total of 17 points, with a median score of 6. Only 8 out of the 54 articles [34,40,44,45,64,66,74,76] had statements about the control of temperature, humidity, and light. 50 articles reported using randomized allocation, none of which reported specific details of randomized allocation method. Only one study [52] reported details of allocation concealment. ree studies [21,52,63] reported using blinded outcome assessment, and 5 studies [32,39,40,46,74] used blinded assessment partly. No study reported using blinded model induction method, blinded intervention administration, or being free of selective reporting. Only 2 articles [48,63] declared sample size calculation or explanation, one [48] of which was hard to evaluate the correctness of calculating method from its statements. 14 studies reported an animal welfare regulation and compliance statement. Only one study [49] did not describe the statistical method. ree articles [21,41,80]

Effects of Acupuncture at ST36 on Cardiac Function in Experimental Sepsis.
Four studies reported that, there were no significant differences in mean arterial blood pressure (MAP) and heart rate (HR) between TGs and the control groups (CGs), whether it was monitored by a small animal tail-cuff blood pressure analyzer [51,52] or by an arterial catheter inserted into the artery [49,63] in rats. However, in another research with rabbits [39], there was significant improvement in MAP and HR when comparing TG with CG. One study [65] reported acupuncture at ST36 significantly improved left ventricular end diastolic pressure (LVEDP), left ventricular end systolic pressure (LVSP), left ventricular pressure maximum rising rate (dp/dt max), cardiac output (CO), and HR, determined by the left cardiac catheterization from the pulmonary vein. Song et al. [68] and Zhang et al. [79] reported that acupuncture therapy could prevent myocardial damage during sepsis by detecting the plasma activity of creatine kinase-MB (CK-MB). EA at ST36 could also significantly lowered the ratios of the water content of heart tissue, indicating the potential of reducing inflammation and edema [68], e effects of upregulating the expression of heme oxygenase-1(HO-1) protein and mRNA in heart tissue might be the mechanism of the protective effect of inhibiting inflammation [35].

Effects of Acupuncture at ST36 Mitigate Damaging
Other Organ Systems in Experimental Sepsis. Acupuncture at ST36 exerted positive effects on immune barrier by increasing the percentage of T lymphocyte [73] and reducing lymphocyte apoptosis in thymus [58,62] and spleen [57]. It was reported that acupuncture at ST36 significantly decreased alanine aminotransferase (ALT) [54,60,61,68,70] and aspartate aminotransferase (AST) [52,70], attenuating sepsis-induced hepatic injury by reducing the tissue water content of liver [55,61,68] and suppressing hepatic inflammatory cytokines (iNOS [55,70], MPO [55,60], and TNF-α [55] [71], demonstrating acupuncture at ST36's potential of reducing inflammation in sepsis. Also, acupuncture could play a positive role in neurotrophy, particularly in the hippocampus, by upregulating Ghrelin, which is essential for cognitive adaptation to changing environments and the process of learning [66,76]. Chen et al. reported that acupuncture at ST36 significantly reduced expression of TLR4 and NF-κB, suggesting a possible pathway involving TLR4 and NF-κB in sepsis animals [74,77] might be the underlying mechanism of acupuncture's beneficial role. Acupuncture at ST36 might play a protective effect on maintaining immune balance in sepsis animals by upregulating CA3+, CD4+, and CD8+ lymphocytes expression and restoring an approximately average level of CD4+/CD8+ ratio [73]. Rafael reported that electroacupuncture at ST36 could control systemic inflammation by inducing a vagal activation of dopamine decarboxylase, thus leading to the production of dopamine in the adrenal medulla, while dopamine inhibits cytokine production via dopaminergic type-1 receptors [21].
Outcome measures of the included studies are summarized in Table 3.

Discussion
To our knowledge, this is the first systematic review of acupuncture at ST36 for sepsis animal model of literatures in both English and Chinese. e present study indicated that acupuncture at ST36 could be useful in reducing sepsisinduced injuries in heat, lung, kidney, liver, gastrointestinal tract, and immune system. Moreover, its potential mechanisms for antisepsis might include decreasing oxidative stress and inflammation, improving microcirculatory disturbance, and maintaining immune balance during sepsis.
However, the heterogeneity among the included studies was significant, the effectiveness of acupuncture therapy might be affected. From the heterogeneity analysis, we can conclude the following implications for further research.

4.1.
Firstly, the Heterogeneity of Acupuncture erapy was One of the Most Critical Factors. Only 2 of the 54 studies conducted MA, and remaining 52 studies performed EA. According to TCM theory, acupuncture produces therapeutic effects by the retention of needles at acupoints through acquiring "Deqi" manually. Deqi is a specific needle sensation, referring to the response to stimulations such as the thrusting, lifting, or rotating of the needle after insertion. It has been asserted to be a criterion to determine the appropriateness of acupuncture stimulation [82,83]. e physical stimulation of MA is the key factor of its effects. However, when it comes to EA, the influence factors will be complicated by electrical stimulation. It is reported that simple focal electrical stimulation on mouse colon, even without acupuncture needle insertion, could produce phased-locked calcium signals in myenteric neurons and produced colon contractions to improve gastrointestinal function [84]. Also, Rafael et al. demonstrated EA at the sciatic nerve controls systemic inflammation by inducing a vagal activation of dopamine decarboxylase, while EA with a wood toothpick did not inhibit cytokine levels [21]. e results mentioned above indicate that only electrical stimulation itself might have a therapeutic effect already. Furthermore, the EA parameters, including waveform, frequency, current, and the pulse width, which differed a lot in the included studies, can be an essential factor influencing EA efficacy and its mechanism [85][86][87]. At the same time, 48.1% (26/54) of the studies used ST36 plus other acupoints. ST36, based on the TCM theory, could harmonize the spleen and stomach, tonify and replenish the middle qi, unblock the meridian and free the collateral vessels, disperse wind and transform dampness, reinforce the healthy qi, and eliminate the pathogenic factors. Adding other acupoints, in TCM theory, could synergize to protect the multiple organs and systems, such as cardiac, brain, lungs, kidneys, and liver. However, whether it is a synergistic interaction or an antagonistic effect actually is still unclear. erefore, further study of acupuncture should pay attention to exploring the appropriate acupuncture intervention and EA parameters to achieve the best efficiency according to different targets.

Secondly, the Heterogeneity of Sepsis Animal Models was Another Factor.
e animal model for sepsis in the included studies mainly lies in the LPS injection model and CLP model. LPS and CLP models could have similar mortality but significant differences in the kinetics and magnitude of cytokine production. LPS injection model is notable for the advantages of technical simplicity and high reproducibility, particularly in the eliciting of
Besides, some neotype sepsis models to compensate the weaknesses of conventional sepsis model has been invented for better reproducing various sepsis physiological progressions [95,96]. So further studies for experimental sepsis should take these factors into consideration and choose the most appropriate model.

irdly, the Quality of Study Design and Reporting Should be Optimized.
e methodological quality of the included studies was generally low. Statement of animal welfare compliance and sample size calculation are essential factors for evaluating the quality of evidence [25], although they are not criteria for risk of bias in animal trials. Most of the included studies had no report of allocation to model induction, intervention administration, blinding of outcome assessors, and outcome assessment and, as a result, risk of observer bias may exist [97].
Moreover, there are several limitations in this systematic review. First, we included literatures published in Chinese or English only in this systematic review, so selective bias might exist. Second, most of the studies were published articles (47 out of 54 studies). Few data were collected from unpublished source. As a result, the efficacy of acupuncture therapy might be overestimated due to publication bias. ird, the general methodological quality of included studies was poor, suggesting that the results should be interpreted with caution.

Conclusions
54 studies were identified from 7 databases in this systematic review to evaluate the efficacy of acupuncture at ST36 for sepsis. In experimental sepsis, acupuncture at ST36 has been reported to be effective in ameliorating systematic injuries induced by sepsis. Poor methodological quality and publication bias exist. Interpreting the positive results should be done carefully. As the use of acupuncture is endorsed by the National Institute of Health and the World Health Organization, acupuncture might be a supplementary strategy for systematic inflammation control. It is worthy of further clinical and experimental trials.