Preclinical Evidence and Mechanism of Xingnaojing Injection for Cerebral Ischemia: A Systematic Review and Meta-Analysis of Animal Studies

Objectives Cerebral ischemia can cause severe harm to people's health with the characteristics of high incidence, high disability, and high mortality. Xingnaojing injection (XNJI) is widely used in the treatment of cerebral ischemia. The aim of this review is to evaluate the efficacy and mechanism of XNJI in animal models of cerebral ischemia. Methods Total seven electronic databases in English or Chinese (CNKI, Wanfang, VMIS, PubMed, MEDLINE, Embase, and the Cochrane Library) about most experiments and studies which came out before June 2018 of XNJI for cerebral ischemia have been searched. Data extraction, quality assessment, and meta-analysis are conducted according to the Cochrane standards and RevMan 5.3 software. Results We have identified 23 eligible studies and made a meta-analysis based on these studies. Meta-analysis shows that XNJI contributes significantly to reduction in neurological deficit score (P = 0.0002, MD = −1.25, 95% CI: −1.92, −0.58) compared with the control group of cerebral ischemia. Subgroup analytic results demonstrate that XNJI has been more effective in animal model of cerebral ischemia-reperfusion injury (P = 0.009, MD = −1.35, 95%CI: −2.36, −0.34) than that of permanent cerebral ischemia (P = 0.0002, MD = −1.08, 95%CI: −1.66, −0.51). Compared with control group, XNJI could remarkably reduce cerebral infarction area (P < 0.00001, MD = −14.98, 95%CI: −21.36, −8.59), brain edema (P < 0.00001, MD = −4.64, 95%CI: −5.38, −3.90), and neuronal cell apoptosis (P < 0.0001, MD = −12.21, 95%CI: 18.05, −6.37). Meanwhile, the meta-analysis shows that XNJI has a significant anti-inflammatory effect, and the levels of TNF-α, IL-6, and IL-1β are significantly reduced by XNJI (P = 0.001, MD = −4.13, 95%CI:−6.68, −1.58; P < 0.00001, MD = −119.23, 95%CI: −138.04, −100.43; P = 0.21, MD = −228.69, 95% CI: −586.20, 128.83). Additionally, XNJI could raise the body's antioxidant function and the level of SOD and GSH-Px (P = 0.002, MD = 53.02, 95% CI: −20.52, 85.78; P = 0.01, MD = 8.65, 95% CI: 1.77, 15.48) and decrease the level of MDA (P < 0.00001, MD = −4.16, 95% CI: −5.50, −2.82). Conclusion XNJI might be effective in cerebral ischemia by regulating oxidative stress and inflammatory reaction.


Introduction
Cerebral ischemia is commonly known as ischemia stroke, accounting for more than 80% of stroke cases. It seriously harms people's health with high incidence, high disability, and high mortality [1,2]. As reported, ischemia stroke is a major cause of global mortality and morbidity [3][4][5]. With the fast pace of modern life and the gradual increase of modern stress, the rate of stroke has increased year by year, and it is tending to encroach on younger adults. Moreover, stroke leads to varying degrees of functional impairment for survivors, which brings serious burden to family and society. It is believed that the increasing global impact of ischemia stroke in the few decades will certainly affect the healthcare in several developing countries, including China.
Cerebral ischemia is caused by cerebral vascular occlusion induced by many reasons. Cerebral vascular occlusion leads to cerebral ischemia, hypoxia, and a series of pathological damage to brain. The physiological of cerebral ischemia is generally thought as a rapid cascade reaction with multilink and multichannel characteristics [6,7]. In this event, the local steady state is broken and the pathological change is produced with the mix of excitotoxicity, oxidative stress, intracellular calcium overload, inflammatory reaction, cellular swelling, and eventually apoptosis or necrosis. In particular, the injury of blood brain barrier (BBB) structure and function is the 2 Evidence-Based Complementary and Alternative Medicine key process. When in reperfusion, inflammatory factors could permeate through the damaged BBB into the brain. Moreover, many of the original harmless substances, such as oxygen molecules, will also be harmful via BBB. These factors lead to brain injury, and the brain injury became more serious during reperfusion. This situation causes great difficulty in the treatment of cerebral ischemia patients [8,9].
Currently, the only effective drug for stroke which has been approved by Food and Drug Administration (FDA) is tissue-type plasminogen activator (t-PA), which is thrombolytic therapy by intravenous injection. It can achieve recanalization of blood flowing that is considered to be the most direct and effective treatment method. However, the drug must be used after a short period of limited stroke medication and secondary risk of bleeding; to a certain extent, it limits the clinical application of thrombolytic therapy [10]. Therefore, the new strategy treatment for stroke as well as long-term brain protection of new drugs is still the field of cerebral ischemia injury prevention and research focus and objectives [11]. And western medicine is increasingly recognized as failing to achieve the desired goals in the treatment of complex diseases, whereas traditional Chinese patent medicine (TCPM) can make substantial improvements in these diseases based on traditional Chinese medicine wholism.
Xingnaojing injection (XNJI), an effective TCPM, is derived from a classic Chinese emergency prescription named An Gong Niu Huang pills. An Gong Niu Huang pills is from "Treatise on Differentiation and Treatment of Epidemic Febrile Diseases" written by Wu Tang in the Qing Dynasty, which is widely used to cure various acute cerebrovascular diseases with good effectiveness. XNJI is produced by the secondary distillation of steam from the following four herbs: artificial Moschus, synthetic Dryobalanops aromatica Gaertn. F., Gardenia jasminoides Ellis, and Radix Curcumae. Moschus. Radix Curcumae. Moschus is the preferred medicine to rescue with aromatics, and combination with Dryobalanops aromatica Gaertn. F. could enhance the effect of resuscitation [2,12]. In recent research, it demonstrated the characteristics and advantages of multitarget, multicomponent and multichannel regulation [12]; XNJI could directly act on the BBB permeability in nerve center by inhibiting inflammatory factors effectively [13][14][15][16][17]. Moreover, it could regulate the effect on cognitive function and antioxidant free radicals [18,19], alleviate encephaledema, and ameliorate the ischemia, anoxic state anticell autophagy, and apoptosis in brain [16,[20][21][22][23][24].
At present, clinical meta-analysis demonstrates that XNJI could promote the recovery of neurological function in patients with cerebral ischemia and reduce the cerebral infarction area [25]. And, there are many studies reporting the mechanism of XNJI on cerebral ischemia; however, there is no publication to summarize the mechanism of Xingnaojing treatment of cerebral ischemia. Therefore, we performed a systematic review and meta-analysis of experimental animal studies to gain a better understanding of the effect and mechanism of XNJI on cerebral ischemia and to explore its mechanism further (Figure 1).

Literature Search.
Literature filtrating was conducted independently by two investigators (Rong Ma and Jianxia Wen), including Chinese National Knowledge Infrastructure (CNKI), Wanfang Database, VIP medicine information system (VMIS), PubMed, MEDLINE, Embase, and the Cochrane Library from the inception to June 2018. The following terms searched were used individually or in combination: "Xingnaojing injection" OR "XNJI" AND "stroke" OR "cerebral ischemia" OR "cerebral ischemia-reperfusion".

Inclusion and Exclusion
Criteria. The inclusion criteria are as follows: (i) the experiment is based on animal model of cerebral ischemia; (ii) treatment group receives the XNJI only; (iii) the included studies contain control group and XNJI treatment group; (iv) studies must include one of the defined outcome measures. The primary outcome measures are as follows: neurological deficit score, brain edema, cerebral infarction area, and neuronal apoptosis; the second outcome measures: tumor necrosis factor-(TNF-), interleukin-6 (IL-6), interleukin-1 (IL-1 ), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA).
The studies were excluded, if they presented the following criteria: (i) clinical studies; (ii) treatment group without XNJI or combined with other agents; (iii) review and/or metaanalysis; (iv) the primary outcome measures and the second outcome measures which are not included in the literature; (v) duplicated publications; (vi) the article which has only an abstract.

Data Collection.
The data extraction and quality assessment of the included studies were conducted independently by two investigators (Rong Ma and Jianxia Wen), and any disagreements were solved through discussion with corresponding author. We have extracted the following data from the included studies: (1) the first author's name and year of publication; (2) species of animals, animal sex, animal weight, numbers of animals in XNJI treatment group, and control group; (3) the model of cerebral ischemia (transient or permanent), the time of cerebral ischemia and reperfusion, XNJI dosage, and intervention duration; (4) primary and second outcome measures. And if the experimental group of animals in the study received various doses of XNJI, only the data of highest dose of XNJI was included.  regulations; (10) statement of potential conflict of interests. Each study was assessed and scored on a scale from 0 (lowest) to 10 (highest) points.

Statistical Analysis.
All values of neurological deficit score, cerebral infarction area, brain edema, neuronal cell apoptosis, and the level of TNF-, IL-6, IL-1 , SOD, GSH-Px, and MDA are considered as continuous data and analysed by RevMan 5.3 (2008 The Nordic Cochrane Centre). Outcomes were presented as mean difference (MD) with 95% confidence interval (CI) and the I-square (I 2 ) statistic used to assess heterogeneity. If the I 2 = 0, which suggested that there is no heterogeneity, fixed effect model would be used to perform a meta-analysis. If the I 2 ≤ 50 % or P ≥ 0.1, it is considered

Records for further screenings (n=225)
Records excluded a er screening based on title and abstract (n=159) Excluded reasons as follows: Full-text articles assessed for eligibility (n=66) minor heterogeneity with fixed effect model. If the I 2 > 50% or P < 0.1, there is significant heterogeneity with random effect model.

Study Characteristics.
A total of 578 animals (290 in the trial group; 288 in the control group) were included in the 23 studies. Sprague-Dawley (SD) rats or Wistar rats were used in most of the animal experiment; other animals such as mongolian gerbil, mice, and rabbits were used in a few experiment. Most of the research projects were male rats, both male and female, were also included. In the selected studies, the experimental animals were predominantly using intravenous or intraperitoneal injection in the dosage of XNJI 0.5 ∼ 50 mL/kg and the same volume of normal saline as a control, and most of the dosing methods were administered in a single dose. Additionally, outcomes included neurological deficit score, brain edema, cerebral infarction area, neuronal apoptosis, serum levels of inflammatory factors (TNF-, IL-6, and IL-1 ), and the antioxidant level of body (SOD, MDA, and GSH-Px). There were also three articles which explored the impact of XNJI on BBB damage [20,29,34]. The detailed characteristics of the 23 articles are shown in Table 1.

Primary Outcome Measures
4.1.1. Neurological Deficit Score. Neurological deficit score was measured in seven studies with 120 animals. Significant heterogeneity occurred in index of neurological deficit score (P < 0.00001, I 2 = 93%). Therefore, random effect model was adopted for meta-analysis. The MD with 95% CI of neurological deficit score was −1.25 (−1.92, −0.58), indicating that XNJI could significantly reduce the neurological deficit score compared with control group (P=0.0002) (Figure 3(a) (Figure 3(b)).

Preclinical Evidence and Mechanism of Xingnaojing Injection for Cerebral Ischemia.
The rapid development of modern technology makes many oral formulations of Chinese medicine developing as injections to meet the needs of modern medicine. XNJI is refined based on the traditional Chinese prescription named An Gong Niu Huang pills [42], which is widely applied in clinic for a variety of coma and cerebrovascular accident patients [43,44]. It could alleviate quickly and effectively the clinical symptoms of cerebral ischemia such as hemiplegia, partial body sensory disorder, aphasia vomiting, and ataxia [45].
The pathogenesis of cerebral ischemia and reperfusion is a rapid cascade reaction. The accumulation of inflammatory cytokines in ischemia tissue is an important factor to aggravate cerebral ischemia and hypoxia. When cerebral ischemiareperfusion occurs, cerebrovascular endothelial cells are activated. Meanwhile, platelets and immune cells release a large number of proinflammatory cytokines, including TNF-×, IL-6, IL-1 , PAF (platelet activating factor), and complement activation products substance. TNF-× is a vital role in the inflammatory network of cytokines and is considered as the trigger medium of a systemic inflammatory reaction [46]. It can directly inhibit vascular endothelial cell function, increase vascular permeability, decrease circulatory resistance, and induce cytokines and adhesion molecules release like the waterfall, resulting in inflammatory damage cascade amplification effect, finally, resulting in wall thickening, stenosis, and cerebral infraction [21,22]. Through the metaanalysis, we found XNJI had significant anti-inflammatory effect. Compared with control group, the TNF-, IL-6, and IL-1 levels of XNJI group were lower by 4.13, 119.23, and 228.69 ng/L ( Figure 5). In addition, the results showed that the I 2 value of IL-6 was 0%, suggesting a relatively reliable therapy.
Oxidative stress is another reaction after cerebral ischemia, and the function of free radical scavenging system in the body decreased during cerebral ischemia. The endogenous antioxidant systems are unbalanced and produced large amounts of free radicals, leading to the peroxidation of lipid, protein and nucleic acid, and the biochemical alteration (SOD ↓, GSH-Px ↓, and MDA↑), and further led to BBB disruption with secondary vasogenic edema, activation of apoptosis, and brain infarction [47,48]. Therefore, the production and release of oxygen free radicals are the key steps that cause cerebral injury. Scavenging oxygen free radicals is an important way to treat cerebral ischemia-reperfusion injury. In theory, block oxidative stress or act on the key molecules could reduce cerebral ischemiareperfusion induced oxidative damage. Our data provide evidence that XNJI could improve the body's antioxidant function after cerebral ischemia-reperfusion ( Figure 6), suggesting that XNJI may reduce the damage of cerebral ischemia by regulating inflammatory and oxidative stress.
The important outcomes of cerebral ischemia and reperfusion injury are neurological deficit score, cerebral infarction area, brain edema, and neuronal cell apoptosis. Inflammatory, oxidative stress, and destroyed BBB could be cause and effect by each other and lead to cerebral edema, cell necrosis and activation of apoptosis [49], and brain infarction, which were manifested as neurological dysfunction in clinic [9]. In the animal experiment, it was tested by neurological deficit score. STAIR (2009) states that [50] multiple indicators should be selected in the study of cerebral ischemia drugs, which should include infarction area and neurological function; this emphasized the importance of behavioral outcomes due to its close relationship to late survival rate clinically. Functional recovery of clinical stroke patients is a major endpoint indicator [51,52]. Evidence showed the beneficial of XNJI on neurological function ( Figure 3) and cerebral infarction area (Figure 4(a)), which is consistent with clinical reports [25]. At the same time, subgroup analysis showed that XNJI had a better effect on the model of cerebral ischemia-reperfusion injury than the model of permanent cerebral ischemia. However, the heterogeneity still existed that may be related to different dose and duration of XNJI. Additionally, there were a study which had reported that XNJI have a significant effect on ultrastructural of brain when ischemia-reperfusion [17] and three studies reported the alleviation of XNJI on BBB damage [20,29,34]; however, we did not conduct a metaanalysis due to its quantitative limitations. The destruction of BBB plays an extremely important role in the process of cerebral ischemia. It is the destruction of BBB that causes the originally innocuous substance into the brain tissue to become harmful substances. Therefore, it is recommended that the BBB research should make a consideration in the further. Moreover, most of the studies included in this study are preventive one-time administration; however, cerebral ischemic stroke is mostly treated with XNJI after onset for one week, clinically. Therefore, it is recommended that study with therapeutic administration and longer administration may be considered in the further.

Strength.
Preclinical efficacy experiments are typically cited to justify the initiation of clinical trials. Our findings contribute to the literature on preclinical design and reinforce our exploratory analysis for the mechanism of XNJI against cerebral ischemia. Moreover, they could eliminate unnecessary repetitive tests and contribute to further study in animal experiments to increase the likelihood of success in future clinical trials. Furthermore, as a traditional Chinese medicine, XNJI is a relatively safe drug. Therefore, it may play a potentially greater role in clinical practice in the future.

Limitations.
The average quality score of studies was 4.5. Many studies have failed to describe their methods in detail, such as randomized trials, blind evaluation of results, and assignment of hidden. Therefore, we recommend that all research published in China should follow a guideline similar to the Consolidated Standards of Reporting Trials (CON-SORT) Statement for clinical studies [53]. These strategies will promote the identification and use of many TCPM (e.g., XNJI) outside China.
In addition, there are some deviations caused by the following reasons: first, different animal species, drug dose, duration of administration, and method of administration

Conclusions
Based on the results of this meta-analysis, the effect of XNJI on cerebral ischemia is encouraging. XNJI may be a promising method to alleviate ischemia-induced brain damage by regulating oxidative stress and inflammatory reaction. Considering being accepted far and wide by practitioners, the experiments with more rigorous experimental design and stronger quality control are required.

Conflicts of Interest
The authors declare that they have no conflicts of interest.