Clinical and Preclinical Systematic Review of Astragalus Membranaceus for Viral Myocarditis

Astragalus membranaceus (AM) is a traditional Chinese medicine, which possesses a variety of biological activities in the cardiovascular systems. We conducted a clinical and preclinical systematic review of 28 randomized clinical control studies with 2522 participants and 16 animal studies with 634 animals to evaluate the efficacy, safety, and possible mechanisms of AM for viral myocarditis (VM). The search strategies were performed in 7 databases from inception to January 2020. Application of the Cochrane Collaboration's tool 7-item checklist, SYRCLE's tool 10-item checklist, and Rev-Man 5.3 software to analyze the risk of bias of studies and data. The results show the score of clinical study quality ranged from 3 to 7 points with an average of 3.32, and the score of animal study quality ranged from 2 to 5 points with an average of 3. In clinical study, AM significantly reduced serum myocardial enzymes and cardiac troponin I levels and improved the clinical treatment efficiency in VM patients compared with the control group (P < 0.05). There was no significant difference in the incidence of adverse reactions (P > 0.05). Significant increase of the survival rate and decrease of the cardiac cardiology score, cardiac enzymes, and cardiac troponin I were compared with the placebo group in animal studies (P < 0.05). The possible mechanisms of AM are largely through antivirus and antivirus receptors, anti-inflammatory, antioxidation, antiapoptotic, antifibrosis, and reducing cardiac calcium load. In conclusion, the findings suggested that AM is a cardioprotection candidate drug for VM.


Introduction
Viral myocarditis (VM) is defined as the inflammatory disease that injured the muscular tissues of the heart, which refers to the pathological lesion including focal or diffuse myocardial cell degeneration and necrosis, interstitial inflammatory cell infiltration, and fibrous exudation caused by viruses [1]. The acute inflammation may develop into subacute and chronic gradually to tissue remodeling, fibrosis, and loss of myocardium architecture and contractile function finally leading the myocarditis of dilated cardiomyopathy (DCM) [2]. It may cause acute heart failure (AHF) and sudden death which is counted at 10% of total sudden death [3].
In addition, the global incidence of myocarditis estimates about 10 to 20 cases per 100 000 of the population, and with the improvement of diagnosis, the prevalence and incidence expected 46% increases in 2030 [4]. According to the causal pathophysiology and clinical symptom of VM, three main treatments including conventional medical treatment, immunomodulatory therapy, and immunosuppressive therapy are used [5]. However, establishing the potential benefits of immunomodulators and antiviral therapy is currently at the preliminary research stage [6]. Although great progress such as intra-aortic balloon pump, ventricular assist device, or extracorporeal membrane oxygenation has been reached in the treatment of cardiac end-point events, the more important goal is to prevent or delay their progress and prevent complications in VM patients [7]. Thus, how to effectively treat VM and prevent AHF has attracted more and more attention to the world.
Astragalus membranaceus (AM) is a famous Qitonifying and immunomodulating herb in traditional Chinese medicine [8]. The main components of AM include flavonoids, saponins, polysaccharides, amino acids, and trace elements [9]. It has been widely used as a natural immunomodulator in the treatment of many immune diseases including nephritis [10], immune reaction of cancer [11], and systemic lupus erythematosus [12], and it also showed efficacy in protecting the myocardium in cardiovascular diseases [13]. In recent years, clinical and basic studies have reported the positive therapeutic effect of AM for VM. However, the scattered clinical evidence and uncertain mechanisms limited the application of AM in the clinic. Therefore, in the present study, we are aimed at comprehensively and systematically evaluating the efficacy, safety, and possible mechanisms of AM for VM from clinical and preclinical aspects.

Data Sources and Search Strategies.
A systematic literature search for the true randomized and controlled studies (RCTs) [14] and animal experimental studies of AM for VM was carried out using PUBMED, EMBASE, Web of Science, Cochrane library, China National Knowledge Infrastructure, Wanfang, and VIP database. All search strategies were performed from inception to January 2020 with the search keyword: "Astragalus" AND "Viral myocardial". Besides, reference lists from the resulting publications and reviews were searched carefully for the potential eligible studies.

Eligibility Criteria.
Two authors selected the studies independently by screening the abstracts and full texts according to the eligibility criteria. Clinical research was included if it met the following criteria: (1) true RCTs of AM for VM with the accepted methodology for randomization: the study which randomized sequence was generated by randomized sequence, calculator, or computer random number generator was included preferentially; coin-tossing or drawing straws in the absence of the participant to decide which group the next participant would be assigned to were also considered eligible randomization techniques [14]; (2) the selected participant should match VM diagnose [2,15,16]; (3) the treatment group involved AM as monotherapy or plus basic treatment with unrestricted dosage, formulation, route of administration, and administration time, and the control group received basic treatment, placebo, basic treatment plus placebo, or no treatment as treatment; (4) the primary outcome measures were mortality or survival rate and/or the main cardiovascular events and/or myocardial enzyme and/or cardiac troponin level and/or the heart function index of ultrasonic cardiogram. We adopted the efficiency of clinical therapy and adverse reaction as the second outcome measures. Animal research was included if it met the following criteria: (1) controlled studies assessing the in vivo administration of AM for VM established by various ways were included; (2) the treatment group involved AM as monotherapy with unrestricted dosage, formulation, route of administration, and administration time, and the control group received placebo or no treatment as treatment; (3) the primary outcome measures were mortality and/or survival rate and/or cardiac pathology and/or myocardial enzyme and cardiac troponin level and/or the heart function index of ultrasonic cardiogram, while the second outcome measures were cardioprotective mechanisms of AM. Exclusion criteria of the clinical and animal researches were as follows: (1) not true RCT study or animal study (in vitro studies, case reports, clinical trials with unaccepted methodology for randomization, reviews, abstracts, comments, and editorials); (2) compare with other Chinese herbals; (3) treatment with AM conjunction with other compounds in animal study; (4) duplicate publications; (5) no any primary outcome indicator were involved or incomplete date; (6) no control group; (7) not VM model.

Data Extraction.
The information were extracted from included studies by two independent authors using a predefined form. Clinical study extracted author, year, the number of participants, ratio of male and female, the therapeutic regimen for treatment and control groups, adverse reaction, and outcome index from each study. Animal study extracted author, years, detail of animals participating in the experiment, the method to induce the model, the therapeutic regimen for treatment and control groups, and outcome index. Only the outcome data of the highest dose group and peak time point group were included. The graph data were measured by Photoshop when the results were only rendered by graphics, and the response was not received from the corresponding authors.

Quality
Estimation of Included Studies. The risk of bias tool recommended by Cochrane Collaboration [17] (The Cochrane Collaboration.http://www.cochrane-handbook .org. (Accessed December 25, 2014)) and SYRCLE's risk of bias tool [18] was adopted separately to estimate the quality of included clinical and animal studies. Disagreements in the process of selecting studies, extracting data, and assessing the quality of studies were resolved by consensus or arbitration by the correspondence authors.

Study Quality.
The number of criteria met in clinical studies varied from 3/7 to 7/7 with the average of 3.32 according to the risk of bias tool recommended by Cochrane Collaboration [17] (The Cochrane Collaboration.http://www .cochrane-handbook.org. (Accessed December 25, 2014)), while the number of criteria met in animal studies varied from 2/10 to 5/10 with an average of 3 according to SYR-CLE's risk of bias tool [18]. Detailed results of methodological quality of clinical and animal studies are presented, respectively, in Tables 5 and 6.

Oxidative Medicine and Cellular Longevity
(2) Effective Rate of Clinical Treatment. The effective rate of clinical treatment was reported in 25 studies [19-23, 25-28, 30-36, 38-46] to contrast the efficacy of AI or AM granule plus basic treatment and basic treatment, except 1 comparative study [37] of AI and placebo. Meta-analysis of the 25 studies showed significant effects of AI plus basic treatment on increasing the effective rate of clinical treatment compared with basic treatment (n = 2245, RR 1.24, 95% CI [1.19 to 1.28], P < 0:00001; heterogeneity: χ 2 = 16:71, df = 24 (P = 0:86); I 2 = 0%, Figure 6). The symmetrical publication bias funnel indicated that there is no obvious publication bias in this study (Figure 7). The remaining 1 study also showed that the efficacy of AI in the treatment of VM was significantly better than that in the placebo group (P < 0:05).

Subgroup
Analysis. The potential confounding factors (including age of animals, varying methods of administration, varying doses of AM, and various durations of treatment) that may increase the heterogeneity of outcome measures were explored using stratified analysis of cardiac pathological score. In the subgroup analysis of age of Balb/c mice, the effect size of the model used mature mice (≥6 weeks) showed better results than immature mice (<6 weeks) (SMD −1.40 vs. SMD −0.97, P = 0:009, Figure 14(d)), and the heterogeneity of two groups decreased obviously. No difference was seen between the intraperitoneal injection group and oral gavage group (SMD −1.01 vs. SMD −1.28, P = 0:06, Figure 14(c)). The heterogeneity of the two groups decreased insignificantly. In the subgroup analysis of durations of treatment, the longer period of AM treatment (>10 days) showed better effect size than the shorter treatment (≤10 days) (SMD −1.28 vs. SMD −1.01, P = 0:05, Figure 14(a)), and the heterogeneity of the longer period group decreased significantly. No difference was seen between the high dose of AM group (≥10 g/kg) and lowdose group (<10 g/kg) (SMD −1.08 vs. SMD −1.15, P = 0:15, Figure 14(b)), and the heterogeneity of two groups decreased insignificantly.

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Oxidative Medicine and Cellular Longevity animals to comprehensively and systematically evaluate the efficacy, safety, and possible mechanisms of AM in the treatment of VM. The quality of the studies included was generally moderate. The evidence available from the present study showed a cardioprotective function of AM for VM animals and patients by multiple mechanisms.

Limitations.
There are some limitations of the present study: (1) English and Chinese literatures were included only in the present study, which may lead to a certain degree of selection bias; (2) All patients were patients with mild viral myocarditis, which may exaggerate the therapeutic effect of AM; (3) clinical adverse reactions were seldom to be reported; (4) most of the included clinical studies are shortterm follow-up studies with small sample size; (5) the studies selected for our analysis had methodological deficiencies, such as seldom using allocation concealment and the blind method.

Implications.
The results of subgroup analysis showed that AM reduced the cardiac pathological score of mature Balb/c mice with VM significantly better than that of immature Balb/c mice (SMD −1.40 vs. SMD −0.97, P = 0:009), which suggests that the age of mice may be the source of high heterogeneity. It may be related to CAR which is the receptor that binds to the Coxsackie virus on cardiomyocytes [63]. The study from Li and Yi showed that the expression of CAR in the myocardium of mice infected with CVB3 increased significantly and reached a peak on the 7th day after infection, and the disease was aggravated simultaneously [55]. However, the expression of CAR decreased significantly after AM treatment [55]. Thus, we draw a conclusion that CAR plays a key role in the process of infection of CVB3 into target cells, and AM was able to downregulate it. Ito et al. [64] found that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats, which is regarded as one of the crucial reasons that CVB3 tends to infect children and causes severe impact. In addition, eliminating CAR was found to prevent signs of inflammatory cardiomyopathy, with essentially no pathology in animal hearts [65]. And the deletion of CAR at the later stage of mice embryo (≥11days) has no effect on the survival   of many embryos to adulthood and heart development [66]. Thus, the development of drugs that inhibit the expression of CAR may be an important direction in the future treatment of VM, especially in children.
The results of another subgroup analysis showed that the longer period of AM treatment (>10 days) showed better effect size than the shorter treatment (≤10 days) (SMD −1.28 vs. SMD −1.01, P = 0:05), which suggests that the duration of treatment may be the source of high heterogeneity. Myocardial injury caused by VM can be subdivided into two stages. In the early few days of the VM, virus replication causes the exposure of intracellular antigens, myocyte necrosis, and activation of the host's immune system. The specific performance is the invasion of NK cells and macrophages followed by T lymphocytes. The subacute stage covers few weeks to several months [7]. It is characterized by activated virus-specific T lymphocytes, which may target the host's organs by molecular mimicry. Two studies [55,59] reported that AM inhibited the replication of CVB3 and directly reduced the cardiac damage caused by viral replication at the acute stage. In addition, AM also inhibited the activation of T lymphocytes by inhibiting the expression of cytokines (TNF-α [49,53,56], IL-8 [52], MCP-1 [51], and MIP-2 [50]) and reducing myocardial injury at the immune reactions stage (subacute stage). The evidences above suggest that long-term (≥10 days) AM treatment may bring greater benefits to VM. However, there are few studies on multiple time points to measure the main outcome indicators at the current stage. Thus, we suggest that further clinical studies or animal experiments could verify the above theory.
The therapeutic effect of myocarditis was significantly related to the severity of the disease. However, in all the animal studies included, no classification of the mice according to the severity of myocarditis was done. Meanwhile, in all the clinical trials, patients were all with mild viral myocarditis, and no deaths were reported. Thus, with the available primary data, it is impossible to do subgroup analysis according to disease severity. We recommend that the severity of myocarditis should be considered and classified in future studies.
It is reported that low-quality trials have a statistically significant 30-50% exaggeration of treatment efficacy compared with high-quality trials [67]. The quality of the included studies in the present study was considered to be moderate to inferior, with 3-7 points for clinical studies, and 2-5 points for animal studies. Most of the studies had methodological deficiencies, such as seldom using allocation concealment and the blind method. In addition, except for the major projects supported by the fund, few studies have registered experiments in advance or published protocols, which may lead to selective reporting bias [68]. Poor experimental design is a major obstacle to translating preclinical animal research into clinical treatments for human diseases [68]. Thus, we recommend that clinical research should refer to the CONSORT (Consolidated Standards of Reporting Trials) 2010 statement [69], animals research should refer to the ARRIVE (The Animal Research: Reporting In Vivo Experiments) guidelines [70], and the use of allocation concealment and blinding should pay more attention to both clinical and animal research. Moreover, multiple details related to animal treatment, such as anesthesia, analgesia, nutrition, environment (temperature, humidity), and euthanasia, should be recorded in detail, as the lack of humane treatment for animals may also affect the accuracy of the results [70]. Animal research should be registered prior to its execution in a generally accessible database (http://www.crd.york.ac.uk/ PROSPERO), and clinical research should be registered (http://www.clinicaltrials.com). It allows verification of the predefined study hypothesis and end-points of the study and reduces publication bias [71].

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Oxidative Medicine and Cellular Longevity ETR affinity and reducing the expression of ET-1 and ANP [13]; and (7) inhibiting virus infection and replication by reducing the expression of CAR [55]. The mechanism is summarized in Figure 15.

Conclusion
Our findings indicate that AM exerted cardioprotective function in VM animals and patients largely through antivirus and antivirus receptors, anti-inflammatory, antioxidation, antiapoptotic, antifibrosis, and reducing cardiac calcium load. However, due to methodological deficiencies in the original study, current research results need to be treated with caution, and further evidence from future high-quality clinical and animal studies is needed. In conclusion, AM is a potential cardioprotective candidate in the treatment of VM.