Ligustrazine Exerts Cardioprotection in Animal Models of Myocardial Ischemia/Reperfusion Injury: Preclinical Evidence and Possible Mechanisms

Ligustrazine (Lig) is one of the main effective components of Ligusticum Chuanxiong Hort, which possesses a variety of biological activities in the cardiovascular system. Here, we conducted a preclinical systematic review to investigate the efficacy of Lig for animal models of myocardial ischemia/reperfusion injury and its possible mechanisms. Twenty-five studies involving 556 animals were identified by searching 6 databases from inception to August 2017. The methodological quality was assessed by using Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist. All the data were analyzed using Rev-Man 5.3 software. As a result, the score of study quality ranged from 2 to 6 points. Meta-analyses showed Lig can significantly decrease the myocardial infarct size, cardiac enzymes and troponin compared with control (P < 0.01). The possible mechanisms of Lig for myocardial infarction are antioxidant, anti-inflammatory, anti-apoptosis activities and improving coronary blood flow and myocardial metabolism. In conclusion, the findings indicated that Lig exerts cardio protection through multiple signaling pathways in myocardial ischemia/reperfusion injury.


INTRODUCTION
Myocardial infarction (MI) is one of the most common causes of death and disability worldwide (Mozaffarian et al., 2015). The injuries inflicted on the myocardium during acute MI are the result of two processes: ischemia and subsequent reperfusion [ischemia/reperfusion (I/R) injury] (Ibáñez et al., 2015). In patients with MI, the treatment of choice for reducing acute myocardial ischemic injury and limiting MI size is timely and effective myocardial reperfusion using either thombolytic therapy or primary percutaneous coronary intervention (PPCI) (Hausenloy and Yellon, 2013). However, abrupt restoration therapy of coronary flow can lead to possible result in adverse events (Heusch and Gersh, 2017) such as reversible impairment of myocardial contractility (myocardial stunning), ventricular arrhythmias, and microvascular dysfunction, for which there is still no effective therapy (Hausenloy and Yellon, 2013).Several strategies have been developed to attenuate and/or modulate the extent of the I/R injury associated with cardiopulmonary attack for years (Chun et al., 2011); however, results of clinical trials disappoint us, and there are some large clinical trials evaluating promising interventions from bench to bedside that have just begun (Frank et al., 2012;Schmidt et al., 2015). Thus, it is urgent to seek new cardioprotective strategies to improve myocardial salvage and cardiac function when myocardial I/R injury happen.
Chuanxiong, Rhizoma Ligustici Chuanxiong, sichuan lovage rhizome, the dried rhizomes of Ligusticum chuanxiong Hort., a perennial herbal plant of the Umbelliferae/Apiaceaefamily, has the function of activating blood and promoting Qi, first recorded in the earliest complete Pharmacopoeia of China, Shennong Bencao Jing (Shennong's Classic of Materia Medica) from Warring States Period to Han Dynasty. Qi, an important concept in Huangdi Neijing (Huangdi's Internal Classic) written in AD 206∼221, is of the vital substances to comprise body and is the vital energy to maintain life (vital Qi), whereas the exogenous pathogenic factors and/or endogenous pathological changes in the body leads to varieties of diseases (pathogenic Qi) (Chen and Chen, 1998;Yuan et al., 2013). Thus, to promote Qi is a key treatment method in traditional Chinese medicine. Chuanxiong has been widely used in the treatment of cardiovacular diseases for thousands of years and is still widely used in modern time due to its extensive biological activities (Lu et al., 2015). Ligustrazine (Lig) (Figure 1) is one of the main effective components of Chuanxiong, which exerts potential cardio/cerebrovascular protective effects (Zhang et al., 2014;Xu et al., 2017). Lig have indicated that Lig and its numerous metabolites have outstanding pharmacokinetic characteristics, such as rapid metabolism, broad distribution and no accumulated toxic effect (Zou et al., 2018). In addition, animal models are invaluable tools for enriching our understanding of the mechanisms, etiology and treatment of human diseases (Sena et al., 2014). Preclinical systematic review can evaluate the efficacy of drugs more systematically, establish a test field for further animal experiments, provide reliable information for drug research, and lay a foundation for future clinical research (Sena et al., 2014;Disma et al., 2016;Zhang et al., 2017). Thus, we aim to evaluate the available preclinical evidence and possible mechanisms of Lig on cardioprotection in animal experiments of myocardial I/R injury.

Data Sources and Search Strategy
Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) statement was abided (Stewart et al., 2015). We electronically searched in PubMed, EMBASE, Science Direct, Web of Science, Wanfang data Information Site, Chinese National Knowledge Infrastructure (CNKI), and VIP information database from inception to the end of August, 2017 using the following key words: "ligustrazine (MeSH Terms) OR tetramethylpyrazine (MeSH Terms) OR Ligustrazine (Title/Abstract) OR tetramethylpyrazine (Title/Abstract)" AND "myocardial infarction OR myocardial ischemia OR myocardial ischemia/reperfusion injury OR myocardial I/R injury." Additional studies were identified through the reference lists of relevant reports. All the studies included were limited on animals.

Study Selection and Data Extraction
Two investigators (Zheng Q and Zhu PC) independently screened the titles and/or abstracts, of the search results and assessed the remaining full-text articles for eligibility. Any uncertainty eligibility was resolved by discussion. Studies were eligible for our systematic review if they met: (1) Lig for animal models of myocardial I/R injury established by ligating of the left anterior descending coronary artery (LAD) or injecting intravenously vasoconstrictor; (2) Analyzed interventions was received Lig as monotherapy at any dose. Comparator interventions were isasteric and non-functional liquid (normal saline) or no treatment; (3) the primary outcome measures were MI size and/or cardiac enzymes and/or cardiac troponin T (cTnT) and/or the level of STsegment depression and/or left ventricular ejection fraction (LVEF) and/or shortening fraction (FS). The secondary outcome measures were mechanisms of Lig for myocardial I/R injury. Prespecified exclusion criteria were the treatment of Lig in conjunction with other compounds or Lig-based prescriptions, non-myocardial ischemia model, no control group, not published in peer-review journals, and duplicate publications. In the case of multiple publications from one study, we chose the articles with the largest sample or the earliest publication.
Two independent investigators (Zheng Q and Huang YY) extracted the following details from the included studies: (1) name of first author and year of publication; (2) details (species, number, sex, and weight) of animals for each study; (3) methods to establish animal models of myocardial I/R, and the anesthesia methods for model preparation; (4) the information of treatment group, including therapeutic drug dosage, method of administration, duration of treatment, and the same information of control group; (5) mean value and standard deviation of outcomes. The data of highest dose was selected when the treatment group included various doses of the target drug. The result of the peak time point was included when the data were expressed at different times.

Risk of Bias in Individual Studies
The methodological quality of each included study was evaluated by using Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10item checklist (Macleod et al., 2004) with minor modification (Yu et al., 2017) as follows: A: peer-reviewed publication; B: control of temperature; C: random allocation to treatment or control; D: blinded induction of model; E: blinded assessment of outcome; F: use of anesthetic without significant intrinsic cardioprotective activity; G: appropriate animal model (aged, diabetic, or hypertensive); H: sample size calculation; I: compliance with animal welfare regulations; and J: statement of potential conflict of interests. Every item was given one point. Two investigators (P.C. Z and Y.Y. H) independently evaluated the study quality and divergences were well settled through consulting with correspondence authors.

Statistical Analysis
Meta-analyses and sub-analyses were performed using RevMan V.5.3 software. Outcome measures were all considered as continuous data and given an estimate of the combined overall effect sizes utilizing standard mean difference (SMD) or mean difference (MD) with the effects model. SMD or MD with its 95% confidence interval (CI) was used to assess the strength of efficacy of Lig for myocardial I/R injury. The I 2 statistic was used for assessment of heterogeneity among individual studies. A fixed effects model (I 2 < 50%) or a random effects model (I 2 > 50%) was used depending on the value of I 2 . Probability value p < 0.05 was considered significant.

Summary of Evidence
Twenty-five studies involving 556 animals were identified.
The findings from present study demonstrated that Lig exerts cardioprotection inmyocardial I/R injury, largely through antioxidant, anti-inflammatory, anti-apoptotic activities, and improving coronary blood flow and myocardial metabolism via multiple signaling pathways. Despite significant positive results, we should treat the results consciously because of the flaw of methodological quality.

Limitations
Some limitations should be considered while interpreting this study. First, we only included studies from Chinese and English databases. The absence of studies written in other languages may lead to certain degree of selective bias (Nolting et al., 2012). Second, study quality was considered as moderate, which ranged from 2 to 6 points, may affecting the accuracy of the results (Landis et al., 2012).Third, none of the included studies used animals with relevant comorbidities, which are not in conformity with pathophysiology in patients with MI (Landis et al., 2012). Finally, although the heart protective effect of estrogen has been reported both in clinical and preclinical studies (Menazza et al., 2017), 10 including studies Wang et al., 2005;Li et al., 2006;Xu and Zhang, 2006;Chen et al., 2007;Tang et al., 2007;Yang and Rui, 2007;Zhang et al., 2007;Hu et al., 2008;Shang et al., 2008)adopted female animals in this study.

Implications
Poor design of animal research is considered as a roadblock to translate animal research into promising preclinical drug treatments for human disease (Baginskaite, 2012). In the present study, many domains had flaws in aspects of randomization, allocation concealment, and blinding and sample size calculation, which are the core standards of study design (Moher et al., 2015). A lower-quality study trends toward better outcomes, leading to the global estimated effect overstated (García et al., 2012). Thus, in the future study, we recommended that the experimental research of Lig for MI need be promoted by means of incorporating the ARRIVE guidelines (Kilkenny et al., 2012). The possible mechanisms of Lig for cardioprotective function are summarized as follows: (1) antioxidant through increasing glutathione (GSH) Xu and Zhang, 2006;Hu et al., 2008), and enhancing SOD-induced (Xu et al., 1997;Wan et al., 1998;Xu and Zhang, 2006;Hu et al., 2008;Shang et al., 2008;Gu et al., 2009;Li and Li, 2010;Lv et al., 2012) antioxidant via attenuating chondriokinesis to reduce the release of malondialdehyde (MDA) (Xu et al., 1997;Wan et al., 1998;Xu and Zhang, 2006;Hu et al., 2008;Shang et al., 2008;Gu et al., 2009;Li and Li, 2010;Lv et al., 2012;Zhao et al., 2012); (2) the main anti-inflammatory mechanisms: Lig can inhibit P38MAPK pathway (Qian et al., 2014) and enhance nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/hypoxia-inducible factor 1-alpha (HIF-1α) pathway, and they further inhibited the expression of NF-κB . Simultaneously, Lig can inhibit the expression of the endothelial cell adhesion molecule of E-selectin, P-selectin and intercellular adhesion molecule-1 (Yang et al., 2008). Ultimately,