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 [
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 [
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 [
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
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 [
The meta-analysis process of the literature.
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”.
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-
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 meta-analysis; (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.
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.
The methodological quality of the included studies was assessed based on the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) with 10-item quality checklist [
All values of neurological deficit score, cerebral infarction area, brain edema, neuronal cell apoptosis, and the level of TNF-
A flowchart of the study selection process is shown in Figure
Flow diagram of the systematic review.
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-
The characteristics of the included studies.
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Yan et al. 2017 | Rat/W | M | 250± 20 | 24(12/12) | P | 2/24 | i.p. NS | i.p. XNJ 5 mL·kg−1·d−1(1d) | NDS, NCA |
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Li et al. 2016 | Rat/W | M | NR | 20(10/10) | P | 1.5/72 | i.p. NS | i.p. XNJ 20 mL·kg−1·d−1(7d) | NDS,TNF- |
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Chen et al. 2015 | Rat/W | M | 200~250 | 36(18/18) | P | 0.16/48 | i.p. NS | i.p. XNJ 10 mL·kg−1·d−1(1d) | BE, BBB |
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Wu 2015 | Rat/W | M | 250 ±50 | 24(12/12) | P | 2/70 | i.p. NS | i.p. XNJ 50 mL·kg−1·d−1 (5d) | NDS |
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Zhong et al. 2015 | Rat/W | M | 200~250 | 108(54/54) | P | 0.16/72 | NR | i.p. XNJ 10 mL·kg−1·d−1(3d) | BE, BBB |
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Huang et al. 2014 | Rat/W | M | 280~300 | 20(10/10) | P | 0.3/0.3 | i.p. NS | i.p. XNJ 2 mL·kg−1·d−1(1d) | CIA |
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Ma 2014 | Rat/SD | M | 200 ±20 | 12(6/6) | T | 24/- | i.p. NS | i.p. XNJ 3 mL·kg−1·d−1, 3times (1d) | NDS, GSH-Px |
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Zeng 2013 | Rat/SD | M | 280~300 | 20(10/10) | P | 2/24 | c.i.v NS | c.i.v. XNJ 2 mL·kg−1·d−1(1d) | CIA, NCA |
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Zhu 2012 | Rat/SD | NR | 200 | 40(20/20) | P | 2/168 | c.i.v. NS | c.i.v. XNJ 2 mL·kg−1·d−1(1d) | NDS, CIA, NCA |
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Huang 2012 | Mouse | M | 20~22 | 24(12/12) | P | 2/NR | i.p. NS | i.p. XNJ 10 mL·kg−1·d−1(5d) | CIA, IL-6, IL-1 |
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Li and Xie 2011 | Rat/SD | M | 250~300 | 12(6/6) | P | 2/24 | i.v.NS | i.v. XNJ 10 mL·kg−1·d−1(1d) | NDS, CIA, IL-6, IL-1 |
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Guo et al. 2010 | Rat/SD | M | 200~250 | 16(8/8) | P | 2/24 | i.v.NS | i.v.10 mL·kg−1·d−1(1d) | MDA, SOD, NCA |
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Wu 2007 | Rat/W | M | 260±10 | 43(23/20) | T | 8/- | i.p.NS | i.p. XNJ 8 mL·kg−1·d−1(5d) | TNF- |
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Zhang et al. 2006 | Mouse | M&F | 50-80 | 18(9/9) | P | 0.16/4 | i.v.NS | i.v. XNJ 0.685 mL·kg−1·d−1 (1d) | SOD |
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Lu 2006 | Rat/SD | M | 200~250 | 16(8/8) | P | 2/48 | i.p. NS | i.p. XNJ 10 mL·kg−1·d−1(1d) | SOD, MDA, NCA |
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Shen et al. 2004 | Rat/SD | M | 250 ±20 | 12(6/6) | T | 6/- | NR | i.p. 1.25 mL·kg−1·d−1, 2times(1d) | NDS |
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Wang et al. 2004 | Rabbit | M&F | 2200~3000 | 20(10/10) | P | 0.5/2 | i.v. NS | i.v. XNJI mL·kg−1·d−1(1d) | MDA, SOD |
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Zhou et al. 2002 | Mouse | M | 28~30 | 15(7/8) | P | 0.08/0.16 | NR | i.p. XNJ 5 mL·kg−1·d−1(1d) | GSH-Px, SOD, LMF |
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Wan et al. 2001 | Rat/SD | M&F | 200~250 | 16(8/8) | T | 0.66/- | i.p. NS | i.p. XNJ 3 mL·kg−1·d−1(1d) | GSH-Px |
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Cai et al. 2000 | Rat/W | M | 205±15 | 20(10/10) | T | 3/- | i.p. NS | i.p. XNJ 2 mL·kg−1·d−1(1d) | NS |
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Fu et al. 2000 | Rat/W | M | 200~300 | 30(15/15) | P | 3/3 | NR | i.p. XNJ 20 mL·kg−1·d−1(7d) | BE, CIA, NCA |
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Chen et al 2000 | Rabbit | M&F | 2200~3600 | 20(10/10) | P | 0.5/2 | i.v. NS | i.v. XNJ 1 mL·kg−1(1d) | TNF- |
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Zhang et al 1997 | Rabbit | NR | 2500~3000 | 12(6/6) | P | 0.5/0.5 | NR | i.v. XNJ 40 mL·kg−1(1d) | MDA, GSH-Px, |
Note. XNJI is Xingnaojing injection; SD is Sprague-Dawley; W is Wistar; M is male; F is female; P is permanent; T is transient; i.p. is intraperitoneal injection; i.v. is intravenous injection; c.i.v. is caudal intravenous injection; NR is not report; Index isch/rep(h) is index ischemia and reperfusion in hours; - means no reperfusion; BE is brain edema; BBB is blood brain barrier; NDS is Neurological Deficit Score; CIA is cerebral infarction area; NCA is Neuronal cell apoptosis; TNF-
All studies were published in a peer-reviewed journal. Seven studies described the temperature control [
Quality assessment of included studies.
| | | | | | | | | | | |
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Yan et al. 2017 | √ | UN | √ | UN | UN | UN | √ | √ | UN | UN | 4 |
Li et al. 2016 | √ | UN | √ | UN | UN | √ | √ | √ | UN | UN | 5 |
Chen et al. 2015 | √ | √ | √ | UN | UN | √ | √ | √ | UN | UN | 6 |
Wu 2015 | √ | √ | √ | UN | UN | √ | √ | √ | UN | UN | 6 |
Zhong et al. 2015 | √ | UN | √ | UN | UN | √ | √ | √ | UN | UN | 5 |
Huang et al. 2014 | √ | UN | √ | UN | UN | √ | √ | √ | UN | UN | 5 |
Ma 2014 | √ | √ | √ | UN | UN | √ | √ | UN | UN | UN | 5 |
Zeng 2013 | √ | √ | √ | UN | UN | √ | √ | √ | UN | UN | 6 |
Zhu 2012 | √ | UN | √ | UN | UN | √ | √ | √ | UN | UN | 5 |
Huang 2012 | √ | √ | UN | UN | UN | √ | √ | UN | UN | UN | 4 |
Li and Xie 2011 | √ | UN | √ | UN | UN | UN | √ | √ | UN | UN | 4 |
Guo et al. 2010 | √ | UN | √ | UN | UN | UN | √ | UN | UN | UN | 3 |
Wu 2007 | √ | UN | √ | UN | UN | √ | √ | √ | UN | UN | 5 |
Zhang et al. 2006 | √ | UN | √ | UN | UN | √ | √ | UN | UN | UN | 4 |
Lu 2006 | √ | UN | √ | UN | UN | √ | √ | UN | UN | UN | 4 |
Shen et al. 2004 | √ | √ | UN | UN | UN | UN | √ | UN | UN | UN | 3 |
Wang et al. 2004 | √ | UN | √ | UN | UN | √ | √ | √ | UN | UN | 5 |
Zhou et al. 2002 | √ | UN | √ | UN | UN | √ | √ | UN | UN | UN | 4 |
Wan et al. 2001 | √ | √ | UN | UN | UN | √ | √ | UN | UN | UN | 4 |
Cai et al. 2000 | √ | UN | √ | UN | UN | √ | √ | √ | UN | UN | 5 |
Fu et al. 2000 | √ | UN | √ | UN | UN | UN | √ | √ | UN | UN | 4 |
Chen et al. 2000 | √ | UN | UN | UN | UN | UN | √ | √ | UN | UN | 3 |
Zhang et al. 1997 | √ | UN | UN | UN | UN | √ | √ | √ | UN | UN | 4 |
Note: (1) publication in a peer-reviewed journal; (2) statement of temperature control;(3) random allocation to groups; (4) allocation concealment; (5)blinded assessment of outcome; (6) use of anesthetic without significant internal protection of blood vessel; (7) appropriate animal model (aged, healthy, diabetic, or hypertensive); (8) sample size calculation; (9) compliance with animal welfare regulations; (10) statement of potential conflict of interests. UN is unclear.
Neurological deficit score was measured in seven studies with 120 animals. Significant heterogeneity occurred in index of neurological deficit score (
Forest plot of comparison: (a) neurological deficit score; (b) subgroup of XNJI on transient and permanent ischemia;
Cerebral infarction area, brain edema, and neuronal cell apoptosis were measured in six studies [
Forest plot of comparison: (a) cerebral infarction area; (b) brain edema; (c) neuronal cell apoptosis.
The effect of XNJI on TNF-
Forest plot of comparison: (a) TNF-
SOD, GSH-Px, and MDA level, the direct index of oxidation system of the body, were measured in five [
Forest plot of comparison: (a) SOD; (b) GSH-Px; (c) MDA.
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 [
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 ischemia-reperfusion occurs, cerebrovascular endothelial cells are activated. Meanwhile, platelets and immune cells release a large number of proinflammatory cytokines, including TNF-
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 [
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 [
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.
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 (CONSORT) Statement for clinical studies [
In addition, there are some deviations caused by the following reasons: first, different animal species, drug dose, duration of administration, and method of administration resulted in some deviation. Second, animal models used in most studies are healthy; however, patients with cerebral ischemia are often associated with diabetes, high blood pressure, hyperlipidemia, and the like. Third, our search strategy includes only Chinese and English databases, leading to certain deviations. Thus, the results should be interpreted with caution.
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.
Xingnaojing injection
Traditional Chinese patent medicine
Chinese National Knowledge Infrastructure
VIP medicine information system
Animal Research: Reporting of In Vivo Experiments
Mean difference
Confidence intervals
Consolidated Standards of Reporting Trials
Platelet activating factor
Sprague-Dawley
Wistar
Male
Female
Permanent
Transient
Intraperitoneal injection
Intravenous injection
Caudal intravenous injection
Not report
Index ischemia and reperfusion in hours
Brain edema
Blood brain barrier
Neurological deficit score
Cerebral infarction area
Neuronal cell apoptosis
Brain ultrastructural changes
Brain tumor factor
Interleukin-6
Interleukin-1
Superoxide dismutase
Malondialdehyde
Glutathione peroxidase.
The authors declare that they have no conflicts of interest.
This work is financially supported by grants from National Natural Science Foundation of China (81473371 and 81873023) and Sichuan Province clinical Chinese Pharmacy Science and Technology Innovation Youth Team (2017TD0001).