Effect of Astragaloside IV on improving cardiac function in rats with heart failure: a preclinical systematic review and meta-analysis

Background: Astragaloside IV (ASIV) is the primary pharmacologically active compound found in Astragalus propinquus Schischkin, which has potential protective effects on cardiac function. However, there are almost no systematic evaluations of ASIV for the treatment of heart failure (HF). Methods: Preclinical studies published before 27 December 2022, were retrieved from PubMed, Web of Science, MEDLINE, SinoMed, Chinese National Knowledge Infrastructure (CNKI), VIP information database, and Wanfang Data information site. The quality of included research was evaluated using SYRCLE’s RoB tool. Review Manager 5.4.1 was used to perform meta-analyses of the cardiac function parameters and other indicators. Regression analysis was conducted to observe the dose-efficacy relationship. Results: Nineteen studies involving 489 animals were included. Results indicated that compared with the control group, ASIV could enhance cardiac function indicators, including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular pressure change rate (±dp/dtmax), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), heart weight/body weight (HW/BW) and left ventricular weight/body weight (LVW/BW). Furthermore, the regression analysis showed that the treatment of HF with ASIV was dose-dependent. Conclusion: Findings suggest that ASIV can inhibit cardiac hypertrophy by reducing cardiac preload and afterload, thereby protecting cardiac function.


Introduction
Heart failure (HF) is a complex clinical syndrome that develops due to structural or functional damage of ventricular filling or ejection of blood, and it represents an advanced manifestation of various cardiovascular diseases (Heidenreich et al., 2022).The American Heart Association predicts that by 2030, HF will probably affect more than 8 million people over 18 years old in the United States (Heidenreich et al., 2013).In China, epidemiological surveys report a 4.1% ± 0.3% in-hospital mortality rate for HF (Zhang et al., 2017).Mortality and incidence rates increase with age (Huffman et al., 2013), which contributes to a rising economic burden from HF as the population ages (Cook et al., 2014).Despite significant progress in treatments that have improved the survival of HF patients, such as angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, β receptor blockers, coronary arterial blood revascularization, implantable cardioverter-defibrillators, and cardiac resynchronization therapy (Merlo et al., 2014;Vaduganathan et al., 2020), the 5-year mortality rate of HF remains high (Gerber et al., 2015).
Recent clinical studies suggest that natural medicine could significantly improve the prognosis of HF patients (Wang et al., 2018;Mao et al., 2020;Leung et al., 2021).Astragalus propinquus Schischkin, widely used in traditional Chinese clinical prescriptions, frequently features in prescriptions for treating HF (Guo et al., 2022).In a recent study, Huangqi injection (with active ingredients derived from Astragalus propinquus Schischkin) demonstrated the ability to improve cardiac function (Cao et al., 2022).Huangqi injection was also found to significantly improve various parameters of echocardiography in rats with heart failure, including LVEF and LVFS (Liu et al., 2018).Recent animal studies have shown that Astragaloside IV (ASIV) (Figure 1), the active ingredient of Astragalus propinquus Schischkin, can protect cardiovascular system (Dong et al., 2017;Liu et al., 2021).Accumulating evidence indicates that ASIV can promote angiogenesis (Wang et al., 2013), protect myocardial cells (Luo et al., 2019), and inhibit ventricular remodeling (Lu et al., 2017).
Preclinical systematic reviews can identify areas for testing in further animal experiments, preclude unnecessary study replication, refine animal experimentation, and lay the foundation for future clinical trials (Murphy and Murphy, 2010).Therefore, in this study, we conducted a systematic review and meta-analysis to evaluate the beneficial effects of ASIV on cardiac function in HF rat models.The results of our study could provide a reference for refining animal experimentation and designing clinical research, as well as identifying new therapeutic strategies for the treatment of HF.

Methods
This systematic review was registered (Invoice Number: CRD42023383485) in PROSPERO (https://www.crd.york.ac.uk/PROSPERO/) and has been reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).

Search strategies
We conducted a comprehensive search of studies on the effect of ASIV in animal models of HF using various electronic databases, such as PubMed, Web of Science, MEDLINE, SinoMed, Chinese National Knowledge Infrastructure (CNKI), VIP information database, and Wanfang Data information site, from their inception to December 2022.

Inclusion/exclusion criteria
To prevent bias, prespecified inclusion criteria were as follows: (1) rat models of HF, without limiting specific modeling method; (2) a controlled experiment; (3)  (2) duplicate publications; (3) Missing result data that can be obtained.

Data extraction
Two authors independently extracted data as follows: 1) the first author's name and publication year; 2) the information of experimental animals such as number, species, sex, weight age; 3) the induction method of HF animal model; 4) the time of experimental drug intervention; 5) the information of treatment used in experimental group such as dose, method of administration, and duration of treatment; 6) the primary outcome measures.If there were multiple measurement results at different times, we recorded the last result.If the experimental animals received different doses of drug intervention, we recorded only the highest dose.The data were measured by the digital ruler software if the data was presented with graphs.For incomplete published data, we contacted the author for further information.For each comparison, we extracted the mean and standard deviation from the experimental and control groups of each study.Discrepancies were resolved after discussion between the two authors.

Outcome
The data for LVEF and LVFS were obtained through echocardiography measurements.The data for LVEDP, LVSP, and ±dp/dt max were obtained through hemodynamic monitoring.The data for HW/BW and LVW/BW were obtained through postmortem measurements and calculations.

Quality assessment
We evaluated the methodological quality of the included studies using the SYRCLE's RoB tool (Hooijmans et al., 2014) with minor modification as follows: 1) randomization of sequence generation; 2) description of baseline characteristics; 3) allocation concealment; 4) animals randomly standardized housed; 5) feeding and intervention in blind; 6) criterion for the success of animal models; 7) random outcome assessment; 8) blinded assessment of outcomes; 9) incomplete outcome data; (10) Other sources of bias.We tried to quantify the evaluation results.Each study was given a total score of ten, with one point for each entry.Two authors independently evaluated the study quality, and disagreement was resolved through discussion or consultation.

Statistical analysis
We performed a meta-analysis using Review Manager 5.4.1.All the data of cardiac function were considered as continuous data, and then, we use mean deviation (MD) and random effect model (REM) to estimate the size of combined effects.Because of the heterogeneity between multiple studies must be considered, in this meta-analysis, we chose the REM to get the results.The χ 2 test with a significance level of = 0.1 will be used as statistical measure of heterogeneity between the different studies.Moreover, the I 2 statistic will be applied to quantifies inconsistency between studies, calculated as I 2 = (Qdf)/ Q*100%, where I 2 statistic of 50% or more indicated a considerable heterogeneity, then additional subgroup and/or sensitivity analysis was performed.Probability values of 0.05 were considered significant.In addition, Origin 2021 was used for dosage-efficacy interval analyses, and regression analysis was used to test the reliability of the dosage-efficacy interval.

LVSP (mmHg)
Eight studies (Zhao et al., 2009;Cui et al., 2013;Zhang et al., 2015;Jiang et al., 2016;Cheng, 2017;Lv, 2018;Zhao et al., 2018;Shi et al., 2021) reported LVSP, and the results of meta-analysis showed that ASIV could not be considered to increase LVSP (n = 214, MD 6.82, 95% CI: −15.47~29.10,p = 0.55; heterogeneity Chi 2 = 975.80,p < 0.01, I 2 = 99%) (Figure 4C).High heterogeneity may be due to different methods of modeling or different anesthetics.Because of high heterogeneity and subgroup analysis and sensitivity analysis cannot reasonably explain the source of heterogeneity, we consider qualitative analysis.Two studies (Jiang et al., 2016;Lv, 2018) reported that ASIV decreased LVSP compared with the control group (p < 0.01).LVSP decreases in HF (Walley, 2016).We noticed that in these two studies, LVSP in the HF model group was higher than that in the sham group.The author did not explain or analyze this in the results.It may be the compensatory increase caused by AAC (Katz et al., 2019).Hence, we consider that it is inappropriate to combine the results of these two studies with other studies After excluding these two studies, the other six studies (Zhao et al., 2009;Cui et al., 2013;Zhang et al., 2015;Cheng, 2017;Zhao et al., 2018;Shi et al., 2021) reported that ASIV had a positive effect on reducing LVSP compared with the control group (p < 0.01 or p < 0.05).

Dosage-efficacy analyses
We explored whether the total dose of ASIV would affect the improvement of cardiac function.For this reason, we selected three main indexes (LVEF, LVEDP and LVW/BW) to evaluate cardiac function and analyzed the dosage-efficacy relationship.First, we excluded the study with extremely low dose (<5 mg/kg/d).For the index of LVEDP, when the total dose of ASIV ranged from 400 mg/kg to 3920 mg/kg, the dosageefficacy relationship shows a significant positive correlation (Significance F < 0.01, p < 0.01).However, it should be noted that for the index of LVEF, the dosage-efficacy relationship did not show a positive correlation at the dose of 700 mg/kg -3360 mg/kg (Significance F > 0.05, p > 0.05).For the index of LVW/BW (mg/g), when the total dose of ASIV ranged from 840 mg/kg to 3920 mg/kg, the dosage-efficacy relationship shows a significant positive correlation (Significance F < 0.01, p < 0.01) (Figure 6).These results may be affected by the mode of model establishment, the drug intervention starting time, the duration of intervention and other factors.Therefore, we consider carefully that in the range of ASIV dosage from 10 mg/kg/d to 80 mg/kg/d, the effect of treating HF may be dose-dependent and/or time-dependent, but this relationship might be nonlinear.

Summary of evidence
Our meta-analysis comprised 19 studies, encompassing a total of 489 animals.Our meta-analysis demonstrates that ASIV exerts cardioprotective effects in HF, as evidenced by increased LVEF, LVFS, and LV ± dp/dt max , as well as decreased LVSP, LVEDP, HW/ BW and LVW/BW.ASIV has been shown to enhance cardiac function post myocardial infarction by inhibiting myocardial fibrosis (Zhang et al., 2022) and promoting angiogenesis (Cheng et al., 2019).Our findings also support this conclusion as evidenced by the changes in the HW/BW and LVW/BW.The dosage-efficacy relationship of ASIV is positively correlated in a range of 10-80 mg/kg/d, indicating that higher doses and longer intervention times may be more effective in treating HF with ASIV, but this relationship may not increase linearly.

Highlights and limitations
This meta-analysis and systematic review evaluated the latest research on the therapeutic effects of ASIV in ameliorating heart function decline caused by HF.In the past 3 years from 2020 to 2022, eight related animal experimental studies have been published.However, there have been no recent studies reviewing and discussing animal experiments, hence our work is timely and necessary.Our study focused on targeted analyses of multiple measurements to assess the positive effects of ASIV on reducing cardiac preload and afterload while inhibiting cardiac hypertrophy under conditions of HF.The dosage-efficacy interval analyses provide valuable information for future animal experiments to determine appropriate treatment times and doses.Additionally, this study contributes to reducing duplicate animal studies, improving animal research design, and provides reference evidence for converting preclinical experimental results into clinical use.
Some limitations of the study are listed as follows.The methodological quality of the included studies is generally poor.All studies lacked descriptions of allocation concealment and random placement of animals, and there were no reports of blinding with regard to feeding or intervention.Poor methodological quality is an inherent limitation that can impact  accuracy (Landis et al., 2012).Furthermore, eight studies employed chloral hydrate as an anesthetic agent.Intraperitoneal administration of chloral hydrate in rats can induce nonmechanical intestinal obstruction, peritonitis, gastric ulcers, and intraperitoneal hemorrhage, which raises ethical concerns in animal research (Silverman and Muir, 1993;Baxter et al., 2009;Percie du Sert et al., 2020).Furthermore, chloral hydrate may elicit intricate effects on the cardiovascular system, thereby compromising the reliability of the results (Laurent et al., 2006;Han et al., 2011;Grissinger, 2019).Therefore, due to the imperfections in some experimental designs, we should treat the present positive results with caution.Given that ASIV's effect on treating HF may be multitargeted, additional research is necessary to analyze potential mechanisms of action.Moreover, because of the small sample size, the dosage-efficacy relationship of ASIV in treating HF requires further investigation with larger sample sizes and higher-quality evidence.

Implications
Numerous studies have demonstrated the crucial role of highquality animal experiments as a reference point for drugs in preclinical research prior to clinical trials.However, given the vast differences between animal models and clinical practice, meticulous attention must be paid to the experimental design in preclinical research.This systematic review highlights key considerations for researchers, including the necessity of providing detailed descriptions of baseline characteristics before and after establishing animal models, as well as the use of standardized assessments, such as the SYRCLE Risk of Bias tool and the ten-item scale, to promote methodological quality.In particular, randomization and blinding techniques should be fully employed throughout the experimental process, including during model induction and outcome assessment.In the majority of relevant in vivo investigations, SD rats or Wistar rats are commonly employed as animal models.However, the utilization of genetically modified mice holds paramount significance in elucidating the underlying mechanisms, thereby warranting the recommendation for a more diversified selection of genetically edited mice to explore potential mechanisms.Exploring various administration methods assumes critical importance in attaining a comprehensive understanding of drug delivery efficacy and variations, consequently enriching our overall comprehension of experimental outcomes.In light of this, we recommend including research on different administration routes to address this knowledge gap.Meanwhile, taking into account the ethics of animal experiments and the impact of anesthesia on cardiovascular indicators, we recommend the use of isoflurane or pentobarbital sodium as anesthetic agents.HF typically presents in elderly patients with underlying conditions such as hypertension.Therefore, the use of relevant animal models can enhance the meaningfulness of the results.In the treatment of HF, long-term interventions and therapies are of paramount importance (Arrigo et al., 2020).Therefore, it is equally crucial to enhance our understanding of the enduring impact of ASIV on overall prognosis by increasing relevant research, thus further investigating the clinical prospects of ASIV's application in HF management.We stress the importance of conducting studies with a wider dose range, including grouping doses, to determine the optimal dosing regimen.Such studies are essential to improving the clinical relevance and translatability of experimental results (Singh et al., 2022).

Conclusion
ASIV, a promising natural compound, has garnered significant attention due to its anti-inflammatory, antioxidant stress, neuroprotective, and other beneficial effects (Liang et al., 2023).It has been extensively investigated for its potential therapeutic applications in cardiovascular and cerebrovascular diseases, hepatitis, cancer, and other conditions (Chen et al., 2021;Li et al., 2022).Part of the pharmacological effects of ASIV can be attributed to its hydrolyzed active metabolite, Cycloastragenol (Yu et al., 2018).Regarding pharmacokinetics, ASIV exhibits relatively low bioavailability and absorption rates in the gastrointestinal tract of rats, with an absolute bioavailability of 2.2% (Gu et al., 2004).The elimination half-life of AS-IV in rats ranges from 34.0 to 131.6 min (Zhang et al., 2006).Following intravenous administration, ASIV is rapidly absorbed and widely distributed in various tissues.The kidneys and liver show the highest concentrations of ASIV, followed by the lungs, heart, and spleen.However, ASIV has limited distribution in the brain, likely due to its poor ability to cross the blood-brain barrier (Chang et al., 2012).It is important to note that there is limited research on the drug metabolism and safety of ASIV, and the quality of existing studies is not optimal.This poses a challenge for further exploration of the clinical therapeutic effects of ASIV.Most studies have utilized relatively low dosages and short administration durations, which may not be sufficient to observe acute and chronic toxicity.Therefore, more comprehensive investigations are needed to fully understand the potential benefits and safety profile of ASIV.
Additionally, the lack of high-quality meta-analyses and systematic reviews contributes to a limited understanding of the preclinical research efficacy of ASIV.Our study presents initial preclinical evidence supporting ASIV as a promising drug candidate for HF therapy.ASIV shows potential to safeguard cardiac function by decreasing cardiac preload and afterload, as well as inhibiting myocardial hypertrophy.Notably, our dose-effect analysis indicates that ASIV's therapeutic effects range from 10 mg/kg to 80 mg/kg daily dosage, with a possible non-linear positive relationship between the dose and the efficacy.

FIGURE 2
FIGURE 2Flow diagram.The process of papers inclusion was divided into three steps: search, deduplication, and manual screening.Only literature that met the inclusion criteria were included.
FIGURE 3 (A) The forest plot: subgroup analysis of ASIV in sham group (LVEF <70%), sham group (70% < LVEF <90%) and sham group (LVEF >90%) for improving LVEF compared with the control group.(B) The forest plot: subgroup analysis of ASIV in prophylactic administration group, acute phase administration group, chronic phase administration group and not mentioned group for improving LVEF compared with the control group.(C) Funnel plot indicating a predominantly symmetrical distribution of the 11 included studies assessing the outcome of LVEF.(D) The forest plot: effects of ASIV for increasing LVFS compared with the control group.

FIGURE 4 (
FIGURE 4 (A) The forest plot: effects of ASIV for increasing LV + dp/dt compared with the control group.(B) The forest plot: effects of ASIV for increasing LVdp/dt compared with the control group.(C) The forest plot: effects of ASIV for increasing LVSP compared with the control group.(D) The forest plot: effects of ASIV for increasing LVEDP compared with the control group.
FIGURE 5 (A) The forest plot: effects of ASIV for reducing HW/BW compared with the control group.(B) The forest plot: effects of ASIV for reducing LVW/BW compared with the control group.

FIGURE 6
FIGURE 6 Three-dimensional images based on dosage -efficacy interval analyses [(A): LVEF; (B) LVEDP; (C) LVW/BW].The effects of ASIV on improving LVEF was not significant with the increase of dosage.The effects of ASIV on improving LVEDP and LVW/BW were enhanced with the increase of dosage.

TABLE 1
Characteristics of the included studies.

TABLE 1 (
Continued) Characteristics of the included studies.

TABLE 1 (
Continued) Characteristics of the included studies.

TABLE 2
The quality of included studies.